Antibody drug conjugates comprising ecteinascidin derivatives

ABSTRACT

Drug conjugates having formula [D-(X) 6 -(AA) w -(T) g -(L)-] n -Ab wherein: D is a drug moiety having the following formula (I) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, (I) wherein D is covalently attached via a hydroxy or amine group to (X) b  if any, or (AA) w  if any, or to (T) g  if any, or (L); that are useful in the treatment of cancer.

International patent application number PCT/EP2018/060868 is directed tonovel ecteinascidin derivatives which demonstrate very promisinganti-tumor activity. One of the compounds disclosed in such patentapplication is currently in Phase I clinical trials for the preventionand treatment of solid tumors.

FIELD OF THE INVENTION

The present invention relates to novel drug conjugates, drug linkercompounds, to methods for their preparation, pharmaceutical compositionscontaining said drug conjugates and their use as antitumoral agents.

BACKGROUND TO THE INVENTION

The treatment of cancer has progressed significantly in recent yearswith the development of pharmaceutical entities that target and killcancer cells more efficiently. Researchers have taken advantage ofcell-surface receptors and antigens selectively expressed by targetcells such as cancer cells to develop pharmaceutical entities based onantibodies that bind, in the example of tumors, the tumor-specific ortumor-associated antigens. In order to achieve this, cytotoxic moleculessuch as chemotherapeutic drugs, bacteria and plant toxins andradionuclides have been chemically linked to monoclonal antibodies thatbind tumor-specific or tumor-associated cell surface antigens

ADCs therefore represent a challenging area of development given thecomplex payload, linker and antibody structure but there remains a needfor further ADCs to be developed.

SUMMARY OF THE INVENTION

There is a need for novel active drug conjugates. The present inventionaddresses this need. It further provides novel drug linker compounds foruse in the preparation of drug conjugates of the present invention,processes for the preparation of the novel drug conjugates of thepresent invention, pharmaceutical compositions containing said drugconjugates and their use as antitumoral agents, as well as a kitcomprising the drug conjugate of the present invention for use in thetreatment of cancer.

In a first aspect of the present invention there is provided a drugconjugate comprising a drug moiety covalently attached to the rest ofthe drug conjugate, the drug conjugate having formula[D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab wherein:

D is a drug moiety having the following formula (I) or apharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof,

wherein:D is covalently attached via a hydroxy or amine group to (X)_(b) if any,or (AA)_(w) if any, or to (T)_(g) if any, or (L);

Y is —NH— or —O—; R₁ is —OH or —CN;

R₂ is a —C(═O)R_(a) group;R₃ is hydrogen or a —OR_(b) group;R₄ is selected from hydrogen, —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH);R_(a) is selected from hydrogen, substituted or unsubstituted C₁-C₁₂alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(b) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(c) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl; andProt^(NH) is a protecting group for amino,with the optional proviso that when R₄ is hydrogen then Y is —O—;X and T are extending groups that may be the same or different;each AA is independently an amino acid unit;L is a linker group;w is an integer ranging from 0 to 12;b is an integer of 0 or 1;g is an integer of 0 or 1;Ab is a moiety comprising at least one antigen binding site; andn is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] to themoiety comprising at least one antigen binding site and is in the rangefrom 1 to 20.

In a further aspect of the present invention there is provided a drugconjugate comprising a drug moiety covalently attached to the rest ofthe drug conjugate, the compound having formula[D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab wherein:

D is a drug moiety having the following formula (IH) or apharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof,

wherein:the wavy line indicates the point of covalent attachment to (X)_(b) ifany, or (AA)_(w) if any, or to (T)_(g)if any, or to (L);each of Y and Z is independently selected from —NH— and —O—;

R₁ is —OH or —CN;

R₂ is a —C(═O)R_(a) group;R₃ is hydrogen or a —OR_(b) group;R_(a) is selected from hydrogen, substituted or unsubstituted C₁-C₁₂alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl, wherein the optional substituents are oneor more substituents R_(x);R_(b) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl, wherein the optional substituents are oneor more substituents R_(x);substituents R_(x) are selected from the group consisting of C₁-C₁₂alkyl groups which may be optionally substituted with at least one groupR_(y), C₂-C₁₂ alkenyl groups which may be optionally substituted with atleast one group R_(y), C₂-C₁₂ alkynyl groups which may be optionallysubstituted with at least one group R_(y), halogen atoms, oxo groups,thio groups, cyano groups, nitro groups, OR_(y), OCOR_(y), OCOOR_(y),COR_(y), COOR_(y), OCONR_(y)R_(z), CONR_(y)R_(z), S(O)R_(y), SO₂R_(y),P(O)(R_(y))OR_(z), NR_(y)R_(z), NR_(y)COR_(z), NR_(y)C(═O)NR_(y)R_(z),NR_(y)C(═NR_(y))NR_(y)R_(z), aryl groups having from 6 to 18 carbonatoms in one or more rings which may optionally be substituted with oneor more substituents which may be the same or different selected fromthe group consisting of R_(y), OR_(y), OCOR_(y), OCOOR_(y), NR_(y)R_(z),NR_(y)COR_(z), and NR_(y)C(═NR_(y))NR_(y)R_(z), aralkyl groupscomprising an alkyl group having from 1 to 12 carbon atoms substitutedwith an optionally substituted aryl group as defined above, aralkyloxygroups comprising an alkoxy group having from 1 to 12 carbon atomssubstituted with an optionally substituted aryl group as defined above,and a 5- to 14-membered saturated or unsaturated heterocyclic grouphaving one or more rings and comprising at least one oxygen, nitrogen orsulphur atom in said ring(s), said heterocyclic group optionally beingsubstituted with one or more substituents R_(y), and where there is morethan one optional substituents on any given group the optionalsubstituents R_(y) may be the same or different;each R_(y) and R_(z) is independently selected from the group consistingof hydrogen, C₁-C₁₂ alkyl groups, C₁-C₁₂ alkyl groups that aresubstituted with at least one halogen atom, aralkyl groups comprising aC₁-C₁₂ alkyl group that is substituted with an aryl group having from 6to 18 carbon atoms in one or more rings and heterocycloalkyl groupscomprising a C₁-C₁₂ alkyl group that is substituted with a 5- to14-membered saturated or unsaturated heterocyclic group having one ormore rings and comprising at least one oxygen, nitrogen or sulphur atomin said ring(s);X and T are extending groups that may be the same or different;each AA is independently an amino acid unit;L is a linker group;w is an integer ranging from 0 to 12;b is an integer of 0 or 1;g is an integer of 0 or 1;where b+g+w is optionally not 0;Ab is a moiety comprising at least one antigen binding site; andn is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] to themoiety comprising at least one antigen binding site and is in the rangefrom 1 to 20.

In a further aspect of the present invention there is provided a drugconjugate comprising a drug moiety covalently attached to the rest ofthe drug conjugate, the drug conjugate having formula[D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab wherein:

D is a drug moiety having the following formula (I) or apharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof,

wherein:D is covalently attached via a hydroxy or amine group to (X)_(b) if any,or (AA)_(w) if any, or to (T)_(g) if any, or (L);

Y is —NH— or —O—; R₁ is —OH or —CN;

R₂ is a-C(═O)R_(a) group;R₃ is hydrogen or a —OR_(b) group;R₄ is selected from hydrogen, —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH);R_(a) is selected from hydrogen, substituted or unsubstituted C₁-C₁₂alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(b) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(c) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl; andProt^(NH) is a protecting group for amino,with the optional proviso that when R₄ is hydrogen then Y is —O—;X and T are extending groups that may be the same or different;each AA is independently an amino acid unit;L is a linker group;w is an integer ranging from 0 to 12;b is 1;g is an integer of 0 or 1;Ab is a moiety comprising at least one antigen binding site; andn is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] to themoiety comprising at least one antigen binding site and is in the rangefrom 1 to 20.

In a further aspect of the present invention there is provided a drugconjugate comprising a drug moiety covalently attached to the rest ofthe drug conjugate, the drug conjugate having formula[D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab wherein:

D is a drug moiety having the following formula (I) or apharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof,

wherein:D is covalently attached via a hydroxy or amine group to (X)_(b) if any,or (AA)_(w) if any, or to (T)_(g) if any, or (L);

Y is —NH— or —O—; R₁ is —OH or —CN;

R₂ is a-C(═O)R_(a) group;R₃ is hydrogen or a —OR_(b) group;R₄ is selected from hydrogen, —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH);R_(a) is selected from hydrogen, substituted or unsubstituted C₁-C₁₂alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(b) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(c) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl; andProt^(NH) is a protecting group for amino,with the optional proviso that when R₄ is hydrogen then Y is —O—;X and T are extending groups that may be the same or different;each AA is independently an amino acid unit;L is a linker group;w is 2;b is 1;g is an integer of 0 or 1;Ab is a moiety comprising at least one antigen binding site; andn is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] to themoiety comprising at least one antigen binding site and is in the rangefrom 1 to 20.

As we shall explain and exemplify in greater detail below, the drugconjugates of formula [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab of thepresent invention represent a breakthrough in addressing the problemsoutlined above of requiring further drug conjugates in addition to thosebased on the three main families of cytotoxic drugs that have been usedas payloads to date, that show excellent antitumor activity.

In preferred embodiments of the present invention, there is provided adrug conjugate as defined herein, or a pharmaceutically acceptable salt,ester, solvate, tautomer or stereoisomer thereof, wherein D is a drugmoiety selected from formulas (IHa) and (IHb):

Where the wavy lines, R₁, R₂, R₃, Y, and Z are as defined for formula(IH).In a further aspect of the present invention, there is provided acompound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ or of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-H, wherein:L₁ is a linker selected from the group of formulas consisting of:

each of the the wavy lines indicates the point of covalent attachment to(T)_(g) if any, or (AA)_(w) if any, or to (X)_(b) if any, or to D;G is selected from halo, —O-mesyl and —O-tosyl;J is selected from halo, hydroxy, —N-succinimidoxy, —O-(4-nitrophenyl),—O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)—OR₂₀;R₁₉ is selected from —C₁-C₁₂ alkylene-, —C₃-C₈ carbocyclo, —O—(C₁-C₁₂alkylene), —C₆-C₁₈ arylene in one or more rings which may optionally besubstituted with one or more substituents R_(x), —C₁-C₁₂ alkylene-C₆-C₁₈arylene- wherein the arylene group is in one or more rings which mayoptionally be substituted with one or more substituents R_(x), —C₆-C₁₈arylene-C₁-C₁₂ alkylene- wherein the arylene group is in one or morerings which may optionally be substituted with one or more substituentsR_(x), —C₁-C₁₂ alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈ carbocyclo)-C₁-C₁₂alkylene-, —C₅-C₁₄ heterocyclo- wherein said heterocyclo group may be asaturated or unsaturated group having one or more rings and comprisingat least one oxygen, nitrogen or sulphur atom in said ring(s), saidgroup optionally being substituted with one or more substituents R_(x),—C₁-C₁₂ alkylene-(C₅-C₁₄ heterocyclo)- wherein said heterocyclo groupmay be a saturated or unsaturated group having one or more rings andcomprising at least one oxygen, nitrogen or sulphur atom in saidring(s), said group optionally being substituted with one or moresubstituents R_(x), —(C₅-C₁₄ heterocyclo)-C₁-C₁₂ alkylene-, wherein saidheterocyclo group may be a saturated or unsaturated group having one ormore rings and comprising at least one oxygen, nitrogen or sulphur atomin said ring(s), said group optionally being substituted with one ormore substituents R_(x), —(OCH₂CH₂)_(r) and —CH₂—(OCH₂CH₂)_(r), whereineach of the above alkylene substituents whether alone or attached toanother moiety the carbon chain may optionally be substituted by one ormore substituents R_(x);R₂₀ is a C₁-C₁₂ alkyl or an aryl group having from 6 to 18 carbon atomsin one or more aromatic rings, said aryl groups optionally beingsubstituted with one or more substituents R_(x);r is an integer ranging from 1-10;g is an integer of 0 or 1;b is an integer of 0 or 1;w is an integer ranging from 0 to 12; andeach of D, R_(x), X, T, and AA is as defined in the first aspect of theinvention.

In preferred embodiments of the present invention, b+g+w is not 0. Infurther embodiments, b+w is not 0. In yet further embodiments, when w isnot 0, then b is 1. In a further embodiment, when w is 0 then b is 1.

In a further aspect of the present invention, there is provided acompound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ or of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-H, or a pharmaceutically acceptable salt,ester, solvate, tautomer or stereoisomer thereof; wherein each of D, X,AA, T, Li, b, g and w are as defined herein; but further wherein if thecompound is a compound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-H thenb+w+g≠0.

In a preferred embodiment according to aspects of the present invention,n is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] to themoiety comprising at least one antigen binding site and is in the rangefrom 1 to 20. In further embodiments n is in the range from 1-12, 1-8,3-8, 3-6, 3-5 or is 1, 2, 3, 4, 5 or 6 preferably, 3, 4 or 5 or 4.

In a further aspect of the present invention, there is provided a drugmoiety D for use in an antibody drug conjugate. In a further aspect ofthe present invention, there is provided a drug moiety D for use as apayload in an antibody drug conjugate. In a further aspect of thepresent invention, there is provided the use of a drug moiety D asdescribed herein, in the manufacture of an antibody drug conjugate.

In a further aspect of the present invention, there is provided a drugconjugate according to the present invention, for use as a medicament.

In a further aspect of the present invention, there is provided a drugconjugate according to the present invention for use in the treatment ofcancer, and more preferably a cancer selected from lung cancer,colorectal cancer, breast cancer, pancreas carcinoma, kidney cancer,leukaemia, multiple myeloma, lymphoma, gastric and ovarian cancer. Mostpreferred cancer is breast cancer.

In a further aspect of the present invention, there is provided apharmaceutical composition comprising a drug conjugate according to thepresent invention and a pharmaceutically acceptable carrier.

In a further aspect of the present invention, there is provided a methodfor the prevention or treatment of cancer, comprising administering aneffective amount of a drug conjugate according to the present inventionto a patient in need thereof. Preferably, the cancer is selected fromlung cancer, colorectal cancer, breast cancer, pancreas carcinoma,kidney cancer, leukaemia, multiple myeloma, lymphoma, gastric andovarian cancer. Most preferred cancer is breast cancer.

In a further aspect of the present invention, there is provided the useof a drug conjugate according to the present invention in thepreparation of a medicament for the treatment of cancer, and morepreferably a cancer selected from lung cancer, colorectal cancer, breastcancer, pancreas carcinoma, kidney cancer, leukaemia, multiple myeloma,lymphoma, gastric and ovarian cancer. Most preferred cancer is breastcancer.

In a further aspect of of the present invention, there is provided a kitcomprising a therapeutically effective amount of a drug conjugateaccording to the present invention and a pharmaceutically acceptablecarrier. The kit is for use in the treatment of cancer, and morepreferably a cancer selected from lung cancer, colorectal cancer, breastcancer, pancreas carcinoma, kidney cancer, leukaemia, multiple myeloma,lymphoma, gastric and ovarian cancer. Most preferred cancer is breastcancer. A kit according to the present invention may comprise atherapeutically effective amount of a drug conjugate according to thepresent invention and, optionally, instructions for use of the drugconjugate in the treatment of cancer, particularly a cancer selectedfrom lung cancer, colorectal cancer, breast cancer, pancreas carcinoma,kidney cancer, leukaemia, multiple myeloma, lymphoma, gastric andovarian cancer; most preferably for use of the drug conjugate in thetreatment of breast cancer.

In a further aspect of the present invention there is provided a processfor the preparation of a drug conjugate according to the presentinvention comprising conjugating a moiety Ab comprising at least oneantigen binding site and a drug D, Ab and D being as defined herein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following apply to all aspects of the present invention:

In the compounds of the present invention, the alkyl groups may bebranched or unbranched, and preferably have from 1 to about 12 carbonatoms. One more preferred class of alkyl groups has from 1 to about 6carbon atoms. Even more preferred are alkyl groups having 1, 2, 3 or 4carbon atoms. Methyl, ethyl, n-propyl, isopropyl and butyl, includingn-butyl, isobutyl, sec-butyl and tert-butyl are particularly preferredalkyl groups in the compounds of the present invention.

In the compounds of the present invention, the alkenyl groups may bebranched or unbranched, have one or more double bonds and from 2 toabout 12 carbon atoms. One more preferred class of alkenyl groups hasfrom 2 to about 6 carbon atoms. Even more preferred are alkenyl groupshaving 2, 3 or 4 carbon atoms. Ethenyl, 1-propenyl, 2-propenyl,1-methylethenyl, 1-butenyl, 2-butenyl, and 3-butenyl are particularlypreferred alkenyl groups in the compounds of the present invention.

In the compounds of the present invention, the alkynyl groups may bebranched or unbranched, have one or more triple bonds and from 2 toabout 12 carbon atoms. One more preferred class of alkynyl groups hasfrom 2 to about 6 carbon atoms. Even more preferred are alkynyl groupshaving 2, 3 or 4 carbon atoms.

Suitable aryl groups in the compounds of the present invention includesingle and multiple ring compounds, including multiple ring compoundsthat contain separate and/or fused aryl groups. Typical aryl groupscontain from 1 to 3 separated and/or fused rings and from 6 to about 18carbon ring atoms. Preferably aryl groups contain from 6 to about 10carbon ring atoms. Specially preferred aryl groups included substitutedor unsubstituted phenyl, substituted or unsubstituted naphthyl,substituted or unsubstituted biphenyl, substituted or unsubstitutedphenanthryl and substituted or unsubstituted anthryl.

Suitable heterocyclic groups include heteroaromatic and heteroalicyclicgroups containing from 1 to 3 separated and/or fused rings and from 5 toabout 18 ring atoms. Preferably heteroaromatic and heteroalicyclicgroups contain from 5 to about 10 ring atoms, most preferably 5, 6, or 7ring atoms. Suitable heteroaromatic groups in the compounds of thepresent invention contain one, two or three heteroatoms selected from N,O or S atoms and include, e.g., coumarinyl including 8-coumarinyl,quinolyl including 8-quinolyl, isoquinolyl, pyridyl, pyrazinyl,pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl,isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl,indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridyl,purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl,cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl and furopyridyl. Suitable heteroalicyclicgroups in the compounds of the present invention contain one, two orthree heteroatoms selected from N, O or S and include, e.g.,pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl,tetrahydrothiopyranyl, piperidyl, morpholinyl, thiomorpholinyl,thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidyl,oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl,1,2,3,6-tetrahydropyridyl, 2-pirrolinyl, 3-pyrrolinyl, indolinyl,2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl,dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl,3-azabicyclo[4.1.0]heptyl, 3H-indolyl, and quinolizinyl.

The groups above mentioned may be substituted at one or more availablepositions by one or more suitable groups such as OR′, =O, SR′, SOR′,SO₂R′, NO₂, NHR′, NR′R′, =N—R′, NHCOR′, N(COR′)₂, NHSO₂R′,NR′C(═NR′)NR′R′, CN, halogen, COR′, COOR′, OCOR′, OCONHR′, OCONR′R′,CONHR′, CONR′R′, protected OH, protected amino, protected SH,substituted or unsubstituted C₁-C₁₂ alkyl, substituted or unsubstitutedC₂-C₁₂ alkenyl, substituted or unsubstituted C₂-C₁₂ alkynyl, substitutedor unsubstituted aryl, and substituted or unsubstituted heterocyclicgroup, where each of the R′ groups is independently selected from thegroup consisting of hydrogen, OH, NO₂, NH₂, SH, CN, halogen, COH,COalkyl, CO₂H, substituted or unsubstituted C₁-C₁₂ alkyl, substituted orunsubstituted C₂-C₁₂ alkenyl, substituted or unsubstituted C₂-C₁₂alkynyl, substituted or unsubstituted aryl, and substituted orunsubstituted heterocyclic group. Where such groups are themselvessubstituted, the substituents may be chosen from the foregoing list. Inaddition, where there are more than one R′ groups on a substituent, eachR′ may be the same or different.

In the compounds for the present invention, the halogen substituentsinclude F, Cl, Br, and I.

More particularly, in the compounds of the present invention, the alkylgroups in the definitions of R₂₀, Fla, Fib, R_(c), Rx, R_(y) and R_(z)may be straight chain or branched alkyl chain groups having from 1 to 12carbon atoms, and they are preferably an alkyl group having from 1 to 6carbon atoms, more preferably a methyl group, an ethyl group or ani-propyl group, and most preferably a methyl group. In the definitionsof M and Q, they may be straight chain or branched alkyl chain groupshaving from 1 to 6 carbon atoms. Methyl, ethyl, n-propyl, isopropyl andbutyl, including n-butyl, isobutyl, sec-butyl and tert-butyl areparticularly preferred alkyl groups in the compounds of the presentinvention.

In the compounds of the present invention, the alkenyl groups in thedefinitions of R_(a), R_(b), R_(c) and R_(x) are branched or unbranched,and may have one or more double bonds and from 2 to 12 carbon atoms.Preferably, they have from 2 to 6 carbon atoms, and more preferably theyare branched or unbranched alkenyl groups having 2, 3 or 4 carbon atoms.Ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl,and 3-butenyl are particularly preferred alkenyl groups in the compoundsof the present invention.

In the compounds of the present invention, the alkynyl group in thedefinitions of R_(a), R_(b), R_(c) and R_(x) are branched or unbranched,and may have one or more triple bonds and from 2 to 12 carbon atoms.Preferably, they have from 2 to 6 carbon atoms, and more preferably theyare branched or unbranched alkynyl groups having 2, 3 or 4 carbon atoms.

In the compounds of the present invention, the halogen substituents inthe definitions of R_(x), R_(y) and R_(z) include F, Cl, Br and I,preferably Cl.

In the compounds of the present invention, the 5- to 14-memberedsaturated or unsaturated heterocyclic group in the definitions of R_(x)is a heterocyclic group having one or more rings, comprising at leastone oxygen, nitrogen or sulphur atom in said ring(s). The heterocyclicgroup is a group which may be a heteroaromatic group or aheteroalicyclic group, the latter of which may be partially unsaturated,both the aromatic and the alicyclic heterocyclic group containing from 1to 3 separated or fused rings. Preferably the heteroaromatic andheteroalicyclic group contain from 5 to 10 ring atoms. Suitableheteroaromatic groups in the compounds of the present invention containone, two or three heteroatoms selected from N, O and S atoms andinclude, for example, quinolyl including 8-quinolyl, isoquinolyl,coumarinyl including 8-coumarinyl, pyridyl, pyrazinyl, pyrazolyl,pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl,triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl,isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridinyl, purinyl,oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl,cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl and furopyridyl. Suitable heteroalicyclicgroups in the compounds of the present invention contain one, two orthree heteroatoms selected from N, O and S atoms and include, forexample, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydrothiopyranyl, piperidyl, morpholinyl,thiomorpholinyl, thioxanyl, piperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl, andquinolizinyl.

In the compounds of the present invention, the aryl group in thedefinition of R_(x) and R₂₀ is a single or multiple ring compound thatcontain separate and/or fused aryl groups and has from 6 to 18 ringatoms and is optionally substituted. Typical aryl groups contain from 1to 3 separated or fused rings. Preferably aryl groups contain from 6 to12 carbon ring atoms. Particularly preferred aryl groups includesubstituted or unsubstituted phenyl, substituted or unsubstitutednaphthyl, substituted or unsubstituted biphenyl, substituted orunsubstituted phenanthryl and substituted or unsubstituted anthryl, andmost preferred substituted or unsubstituted phenyl, wherein thesubstituents are as indicated above.

In the compounds of the present invention, the aralkyl groups in thedefinitions of R_(x), R_(y) and R_(z) comprise an alkyl group as definedand exemplified above which is substituted with one or more aryl groupsas defined and exemplified above. Preferred examples include optionallysubstituted benzyl, optionally substituted phenylethyl and optionallysubstituted naphthylmethyl.

In the compounds of the present invention, the aralkyloxy groups in thedefinitions of R_(x) comprise an alkoxy group having from 1 to 12 carbonatoms which is substituted with one or more aryl groups as defined andexemplified above. Preferably, the alkoxy moiety has from 1 to 6 carbonatoms and the aryl group contains from 6 to about 12 carbon ring atoms,and most preferably the aralkyloxy group is optionally substitutedbenzyloxy, optionally substituted phenylethoxy and optionallysubstituted naphthylmethoxy.

In the compounds of the present invention, the heterocycloalkyl groupsin the definitions of R_(y) and R_(z) comprise an alkyl group as definedand exemplified above which is substituted with one or more heterocyclylgroups as defined and exemplified above. Preferably, theheterocycloalkyl groups comprise an alkyl group having from 1 to 6carbon atoms which is substituted with a heterocyclyl group having from5 to 10 ring atoms in 1 or 2 ring atoms and can be aromatic, partiallysaturated or fully saturated. More preferably, the heterocycloalkylgroups comprise a methyl or ethyl group which is substituted with aheterocyclyl group selected from the group consisting of pyrrolidinyl,imidazolidinyl, piperidinyl, piperazinyl, morpholinyl,tetrahydrofuranyl, oxanyl, thianyl, 8-quinolyl, isoquinolyl, pyridyl,pyrazinyl, pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl,isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl andbenzimidazole.

In the compounds of the present invention, the alkylene groups in thedefinition of R₁₉ are straight or branched alkylene groups having from 1to 12 carbon atoms and the alkylene groups in the definitions of M, X,T, and R₃₀ are straight or branched alkylene groups having from 1 to 6carbon atoms. Preferably, the alkylene groups in the definition of R₁₉are straight or branched alkylene groups having from 1 to 8 carbonatoms, more preferably straight or branched alkylene groups having from1 to 6 carbon atoms. For M, preferred are straight or branched alkylenegroups having from 1 to 3 carbon atoms. In the definition of X, thealkylene groups in the definition of X are preferably straight orbranched alkylene groups having from 2 to 4 carbon atoms. For T,preferred are straight or branched alkylene groups having from 2 to 4carbon atoms. In the definition of R₃₀, preferred are straight orbranched alkylene groups having from 2 to 4 carbon atoms, being mostpreferred a straight alkylene group having 3 carbon atoms. For theavoidance of doubt, the term “alkylene” is used to refer to alkanediylgroups.

In the compounds of the present invention, the carbocyclo groups in thedefinitions of R₁₉ and M are cycloalkyl groups having from 3 to 8 carbonatoms which have two covalent bonds at any position on the cycloalkylring connecting said cycloalkyl group to the remainder of the drugconjugate. Preferably, the carbocyclo groups in the definitions of R₁₉and M are cycloalkyl groups having from 3 to 7 carbon atoms, and morepreferably carbocyclo groups having from 5 to 7 carbon atoms.

In the compounds of the present invention, the arylene groups in thedefinition of R₁₉ are aryl groups having from 6 to 18 carbon atoms inone or more rings which have two covalent bonds at any position on thearomatic ring system connecting said arylene groups to the remainder ofthe drug conjugate. Preferably, the arylene groups in the definition ofR₁₉ are aryl groups having from 6 to 12 carbon atoms in one or morerings which have two covalent bonds at any position on the aromatic ringsystem, and most preferably they are phenylene groups.

In the compounds of the present invention, the heterocyclo groups in thedefinition of R₁₉ are heterocyclyl groups containing from 1 to 3separated or fused rings having from 5 to 14 ring atoms and comprisingat least one oxygen, nitrogen or sulphur atom in said ring(s), whereinthere are two covalent bonds at any position on the ring system of saidheterocyclic groups. The heterocyclic groups are groups which may beheteroaromatic groups or heteroalicyclic groups (the latter may bepartially unsaturated). Preferably, the heterocyclo groups in thedefinition of R₁₉ are heterocyclyl groups containing from 1 to 3separated or fused rings having from 5 to 12 ring atoms and comprisingat least one oxygen, nitrogen or sulphur atom in said ring(s), whereinthere are two covalent bonds at any position on the ring system of saidheterocyclic groups.

Where there are more than one optional substituents R_(x), R_(y) orR_(z) on a substituent, each substituent R_(x) may be the same ordifferent, each substituent R_(y) may be the same or different and eachR_(z) may be the same or different.

In an embodiment, D may be a compound of formula I or a pharmaceuticallyacceptable salt or ester thereof:

wherein:

Y is —NH— or —O—; R₁ is —OH or —CN;

R₂ is a —C(═O)R_(a) group;R₃ is hydrogen or a —OR_(b) group;R₄ is selected from hydrogen, —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH);R_(a) is selected from hydrogen, substituted or unsubstituted C₁-C₁₂alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(b) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(c) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl; andProt^(NH) is a protecting group for amino.

In an embodiment, the compound of formula I has the proviso that when R₄is hydrogen then Y is —O—.

In a further embodiment, the compound of formula I may be a compound offormula IC, or a pharmaceutically acceptable salt or ester thereof:

wherein:

-   -   Y is —NH—;    -   R₁ is —OH or —CN;    -   R₂ is a —C(═O)R_(a) group;    -   R₃ is hydrogen or a —OR_(b) group;    -   R₄ is selected from —CH₂OH, —CH₂O—(C═O)R_(c), —CH₂NH₂ and        —CH₂NHProt^(NH);    -   R_(a) is selected from hydrogen, substituted or unsubstituted        C₁-C₁₂ alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and        substituted or unsubstituted C₂-C₁₂ alkynyl;    -   R_(b) is selected from substituted or unsubstituted C₁-C₁₂        alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl and        substituted or unsubstituted C₂-C₁₂ alkynyl;    -   R_(c) is selected from substituted or unsubstituted C₁-C₁₂        alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and        substituted or unsubstituted C₂-C₁₂ alkynyl; and    -   Prot^(NH) is a protecting group for amino.

In a yet further embodiment, the compound of formula I may be a compoundof formula ID, or a pharmaceutically acceptable salt or ester thereof:

wherein:

-   -   Y is —O—;    -   R₁ is —OH or —CN;    -   R₂ is a —C(═O)R_(a) group;    -   R₃ is hydrogen or a —OR_(b) group;    -   R₄ is selected from hydrogen, —CH₂OH, —CH₂O—(C═O)R_(c), —CH₂NH₂        and —CH₂NHProt^(NH);    -   R_(a) is selected from hydrogen, substituted or unsubstituted        C₁-C₁₂ alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and        substituted or unsubstituted C₂-C₁₂ alkynyl;    -   R_(b) is selected from substituted or unsubstituted C₁-C₁₂        alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl and        substituted or unsubstituted C₂-C₁₂ alkynyl;    -   R_(c) is selected from substituted or unsubstituted C₁-C₁₂        alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and        substituted or unsubstituted C₂-C₁₂ alkynyl; and    -   Prot^(NH) is a protecting group for amino.

In a yet further embodiment, the compound of formula I may be a compoundof formula IE, or a pharmaceutically acceptable salt or ester thereof:

wherein:

-   -   Y is —NH— or —O—;    -   R₁ is —OH or —CN;    -   R₂ is a —C(═O)R_(a) group;    -   R₃ is hydrogen or a —OR_(b) group;    -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH);    -   R_(a) is selected from hydrogen, substituted or unsubstituted        C₁-C₁₂ alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and        substituted or unsubstituted C₂-C₁₂ alkynyl;    -   R_(b) is selected from substituted or unsubstituted C₁-C₁₂        alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl and        substituted or unsubstituted C₂-C₁₂ alkynyl; and    -   Prot^(NH) is a protecting group for amino.

In a yet further embodiment, the compound of formula I may be a compoundof formula IA or a pharmaceutically acceptable salt or ester thereof:

wherein:

-   -   Y is —NH— or —O—;    -   R₁ is —OH or —CN;    -   R₂ is a —C(═O)R_(a) group;    -   R₃ is hydrogen;    -   R₄ is selected from hydrogen, —CH₂OH, —CH₂O—(C═O)R_(c), —CH₂NH₂        and —CH₂NHProt^(NH);    -   R_(a) is selected from hydrogen, substituted or unsubstituted        C₁-C₁₂ alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and        substituted or unsubstituted C₂-C₁₂ alkynyl;    -   R_(c) is selected from substituted or unsubstituted C₁-C₁₂        alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and        substituted or unsubstituted C₂-C₁₂ alkynyl; and    -   Prot^(NH) is a protecting group for amino.        In an embodiment, the compound of formula IA has the proviso        that when R₄ is hydrogen then Y is —O—.

In a yet further embodiment, the compound of formula I may be a compoundof formula IB or a pharmaceutically acceptable salt or ester thereof:

wherein:

-   -   Y is —NH— or —O—;    -   R₁ is —OH or —CN;    -   R₂ is a —C(═O)R_(a) group;    -   R₃ is a —OR_(b) group;    -   R₄ is selected from hydrogen, —CH₂OH, —CH₂O—(C═O)R_(c), —CH₂NH₂        and —CH₂NHProt^(NH);    -   R_(a) is selected from hydrogen, substituted or unsubstituted        C₁-C₁₂ alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and        substituted or unsubstituted C₂-C₁₂ alkynyl;    -   R_(b) is selected from substituted or unsubstituted C₁-C₁₂        alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl and        substituted or unsubstituted C₂-C₁₂ alkynyl;    -   R_(c) is selected from substituted or unsubstituted C₁-C₁₂        alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and        substituted or unsubstituted C₂-C₁₂ alkynyl; and    -   Prot^(NH) is a protecting group for amino.        In an embodiment, the compound of formula IB has the proviso        that when R₄ is hydrogen then Y is —O—.

In a yet further embodiment, the compound of formula I may be a compoundof formula IF or a pharmaceutically acceptable salt or ester thereof:

wherein:

Y is —NH— or —O—; R₁ is —OH;

R₂ is a —C(═O)R_(a) group;R₃ is hydrogen or a —OR_(b) group;R₄ is selected from hydrogen, —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH);R_(a) is selected from hydrogen, substituted or unsubstituted C₁-C₁₂alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(b) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(c) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl; andProt^(NH) is a protecting group for amino.In an embodiment, the compound of formula IF has the proviso that whenR₄ is hydrogen then Y is —O—.

In a yet further embodiment, the compound of formula I may be a compoundof formula IG or a pharmaceutically acceptable salt or ester thereof:

wherein:

Y is —NH— or —O—; R₁ is —OH or —CN;

R₂ is acetyl;R₃ is hydrogen or a —OR_(b) group;R₄ is selected from hydrogen, —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH);R_(b) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl;R_(c) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl; andProt^(NH) is a protecting group for aminoIn an embodiment, the compound of formula IG has the proviso that whenFU is hydrogen then Y is —O—.

Preferred compounds of the compounds of formula I, IA, IB, IC, ID, IE,IF, or IG, are those having general formula a or b, or apharmaceutically acceptable salt or ester thereof:

Note where the compounds have general formula a or b, R₄ may not behydrogen.

Preferred compounds of the compounds of formula I, IA, IB, ID, IF, or IGmay be those having formula c or a pharmaceutically acceptable salt orester thereof:

wherein:R₁ is-OH or —CN;R₂ is a —C(═O)R_(a) group;R₃ is hydrogen or a —OR_(b) group;R_(a) is selected from hydrogen, substituted or unsubstituted C₁-C₁₂alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl; andR_(b) is selected from substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl.For the avoidance of doubt, the compounds above may be the drug moiety Dand are covalently attached via a hydroxy or amine group to (X)_(b) ifany, or (AA)_(w) if any, or to (T)_(g) if any, or (L). Thus, whenconjugated, a covalent bond replaces a proton on a hydroxy or aminegroup on the compound.

Preferred compounds include compounds of general formula I, IA, IB, IE,IF, IG, Ia, IAa, IBa, IEa, IFa, IGa, Ib, IAb, IBb, IEb, IFb, and IGb,wherein:

Y is —NH—;

and R₁; R₂; R₃; R₄; R_(a); R_(b); R_(c); and Prot^(NH) are as defined asabove.

Preferred compounds include compounds of general formula I, IA, IB, IE,IF, IG, Ia, IAa, IBa, IEa, IFa, IGa, Ib, IAb, IBb, IEb, IFb, and IGb,wherein:

Y is —O—;

and R₁; R₂; R₃; R₄; R_(a); R_(b); R_(c); and Prot^(NH) are as defined asabove.

Further preferred compounds include compounds of general formula I, IA,IB, IC, ID, IE, IG, Ia, IAa, IBa, ICa, IDa, IEa, IGa, Ib, IAb, IBb, ICb,IDb, IEb, and IGb, wherein:

R₁ is —OH;

and Y; R₂; R₃; R₄; R_(a); R_(b); R_(c); and Prot^(NH) are as defined asabove.

Further preferred compounds include compounds of general formula I, IA,IB, IC, ID, IE, IF, Ia, IAa, IBa, ICa, IDa, IEa, IFa, Ib, IAb, IBb, ICb,IDb, IEb, and IFb, wherein:

R₂ is a —C(═O)R_(a) group where R_(a) is a substituted or unsubstitutedC₁-C₆ alkyl. Particularly preferred R_(a) is selected from substitutedor unsubstituted methyl, substituted or unsubstituted ethyl, substitutedor unsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl and substituted orunsubstituted tert-butyl. Most preferred R₂ is acetyl;and Y; R₁; R₃; R₄; R_(b); R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IB,IC, ID, IE, IF, IG, Ia, IBa, ICa, IDa, IEa, IFa, IGa, Ib, IBb, ICb, IDb,IEb, IFb, and IGb, wherein:

R₃ is hydrogen or a —OR_(b) group for compounds of formula I, IC, ID,IE, IF, IG, Ia, ICa, IDa, IEa, IFa, IGa, Ib, ICb, IDb, IEb, IFb, or IGb;and R₃ is a —OR_(b) group for compounds of formula IB, IBa or IBb; whereR_(b) is a substituted or unsubstituted C₁-C₆ alkyl. Particularlypreferred R_(b) is selected from substituted or unsubstituted methyl,substituted or unsubstituted ethyl, substituted or unsubstitutedn-propyl, substituted or unsubstituted isopropyl, substituted orunsubstituted n-butyl, substituted or unsubstituted isobutyl,substituted or unsubstituted sec-butyl and substituted or unsubstitutedtert-butyl. More preferred R₃ are hydrogen and methoxy, being hydrogenthe most preferred R₃ group;and Y; R₁; R₂; R₄; R_(a); R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, ID, IE, IF, IG, Ia, IAa, IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb,IBb, ICb, IDb, IEb, IFb, and IGb, wherein:

R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, IC, ID, IF, IG, Ia,IAa, IBa, ICa, IDa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IFb, or IGb; andR₄ is selected from —CH₂NH₂, and —CH₂NHProt^(NH) for compounds offormula IE, IEa or IEb; where R_(c) is a substituted or unsubstitutedC₁-C₆ alkyl. Particularly preferred R_(c) is selected from substitutedor unsubstituted methyl, substituted or unsubstituted ethyl, substitutedor unsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from —CH₂OH and —CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is-CH₂OH;and Y; R₁; R₂; R₃; R_(a); and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—; R₁ is —OH;

and R₂; R₃; R₄; R_(a); R_(b); R_(c); and Prot^(NH) are as defined asabove.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—;

R₂ is a —C(═O)R_(a) for compounds of formula I, IA, IB, IC, IE, IF, Ia,IAa, IBa, ICa, IEa, IFa, Ib, IAb, IBb, ICb, IEb, or IFb; and R₂ isacetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;and R₁; R₃; R₄; R_(b); R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—;

R₃ is hydrogen or a —OR_(b) group for compounds of formula I, IC, IE,IF, IG, Ia, ICa, IEa, IFa, IGa, Ib, ICb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;and R₁; R₂; R₄; R_(a); R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—;

R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa,IBa, ICa, IFa, IGa, Ib, IAb, IBb, ICb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, or substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from CH₂OH and CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH;and R₁; R₂; R₃; R_(a); and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—; R₁ is —OH;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, IC, IE,IF, Ia, IAa, IBa, ICa, IEa, IFa, Ib, IAb, IBb, ICb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;and R₃; R₄; R_(b); R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—; R₁ is —OH;

R₃ is hydrogen or a —OR_(b) group for compounds of formula I, IC, IE,IF, IG, Ia, ICa, IEa, IFa, IGa, Ib, ICb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;and R₂; R₄; R_(a); R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—; R₁ is —OH;

R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa,IBa, ICa, IFa, IGa, Ib, IAb, IBb, ICb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredFU is selected from CH₂OH and CH₂NH₂. More preferably, FU may be—CH₂NH₂. Most preferred FU is —CH₂OH;and R₂; R₃; R_(a); and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is-NH—;R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, IC, IE,IF, Ia, IAa, IBa, ICa, IEa, IFa, Ib, IAb, IBb, ICb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred FU isselected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₃ is hydrogen or a —OR_(b) group for compounds of formula I, IC, IE,IF, IG, Ia, ICa, IEa, IFa, IGa, Ib, ICb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;and R₁; R₄; R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, IC, IE,IF, Ia, IAa, IBa, ICa, IEa, IFa, Ib, IAb, IBb, ICb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa,IBa, ICa, IFa, IGa, Ib, IAb, IBb, ICb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from CH₂OH and CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH;and R₁; R₃; and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—;

R₃ is hydrogen or a —OR_(b) group for compounds of formula I, IC, IE,IF, IG, Ia, ICa, IEa, IFa, IGa, Ib, ICb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa,IBa, ICa, IFa, IGa, Ib, IAb, IBb, ICb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from CH₂OH and CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH;and R₁; R₂; and R_(a); are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—; R₁ is —OH;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, IC, IE,IF, Ia, IAa, IBa, ICa, IEa, IFa, Ib, IAb, IBb, ICb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₃ is hydrogen or a —OR_(b) group for compounds of formula I, IC, IE,IF, IG, Ia, ICa, IEa, IFa, IGa, Ib, ICb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;and R₄; R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—; R₁ is —OH;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, IC, IE,IF, Ia, IAa, IBa, ICa, IEa, IFa, Ib, IAb, IBb, ICb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa,IBa, ICa, IFa, IGa, Ib, IAb, IBb, ICb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from CH₂OH and CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH;and R₃; and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, IC, IE,IF, Ia, IAa, IBa, ICa, IEa, IFa, Ib, IAb, IBb, ICb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₃ is hydrogen or a —OR_(b) group for compounds of formula I, IC, IE,IF, IG, Ia, ICa, IEa, IFa, IGa, Ib, ICb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa,IBa, ICa, IFa, IGa, Ib, IAb, IBb, ICb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredFU is selected from CH₂OH and CH₂NH₂. More preferably, FU may be—CH₂NH₂. Most preferred FU is —CH₂OH;and R₁ is as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, IC, IE, IF, IG, Ia, IAa, IBa, ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb,IEb, IFb, and IGb, wherein:

Y is —NH—; R₁ is —OH;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, IC, IE,IF, Ia, IAa, IBa, ICa, IEa, IFa, Ib, IAb, IBb, ICb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred FU isselected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₃ is hydrogen or a —OR_(b) group for compounds of formula I, IC, IE,IF, IG, Ia, ICa, IEa, IFa, IGa, Ib, ICb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa,IBa, ICa, IFa, IGa, Ib, IAb, IBb, ICb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from CH₂OH and CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—; R₁ is —OH;

and R₂; R₃; R₄; R_(a); R_(b); R_(c); and Prot^(NH) are as defined asabove.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, ID, IE,IF, Ia, IAa, IBa, IDa, IEa, IFa, Ib, IAb, IBb, IDb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;and R₁; R₃; R₄; R_(b); R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—;

R₃ is hydrogen or a —OR_(b) group for compounds of formula I, ID, IE,IF, IG, Ia, IDa, IEa, IFa, IGa, Ib, IDb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃is hydrogen and methoxy, being hydrogen the most preferred R₃ group;and R₁; R₂; R₄; R_(a); R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—;

R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, ID, IF, IG, Ia, IAa,IBa, IDa, IFa, IGa, Ib, IAb, IBb, IDb, IFb, or IGb; and FU is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from —CH₂OH and CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH;and R₁; R₂; R₃; R_(a); and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—; R₁ is —OH;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, ID, IE,IF, Ia, IAa, IBa, IDa, IEa, IFa, Ib, IAb, IBb, IDb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;

-   -   and R₃; R₄; R_(b); R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—; R₁ is —OH;

R₃ is hydrogen or a —OR_(b) group for compounds of formula I, ID, IE,IF, IG, Ia, IDa, IEa, IFa, IGa, Ib, IDb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;and R₂; FU; R_(a); R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—; R₁ is —OH;

R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, ID, IF, IG, Ia, IAa,IBa, IDa, IFa, IGa, Ib, IAb, IBb, IDb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from —CH₂OH and CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH;and R₂; R₃; R_(a); and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, ID, IE,IF, Ia, IAa, IBa, IDa, IEa, IFa, Ib, IAb, IBb, IDb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₃ is hydrogen or a —OR_(b) group for compounds of formula I, ID, IE,IF, IG, Ia, IDa, IEa, IFa, IGa, Ib, IDb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;and R₁; R₄; R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, ID, IE,IF, Ia, IAa, IBa, IDa, IEa, IFa, Ib, IAb, IBb, IDb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, ID, IF, IG, Ia, IAa,IBa, IDa, IFa, IGa, Ib, IAb, IBb, IDb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from —CH₂OH and —CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH;and R₁; R₃; and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—;

R₃ is hydrogen or a —OR_(b) group for compounds of formula I, ID, IE,IF, IG, Ia, IDa, IEa, IFa, IGa, Ib, IDb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, ID, IF, IG, Ia, IAa,IBa, IDa, IFa, IGa, Ib, IAb, IBb, IDb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from —CH₂OH and —CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH;and R₁; R₂; and R_(a); are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—; R₁ is —OH;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, ID, IE,IF, Ia, IAa, IBa, IDa, IEa, IFa, Ib, IAb, IBb, IDb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₃ is hydrogen or a —OR_(b) group for compounds of formula I, ID, IE,IF, IG, Ia, IDa, IEa, IFa, IGa, Ib, IDb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;and FU; R_(c); and Prot^(NH) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—; R₁ is —OH;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, ID, IE,IF, Ia, IAa, IBa, IDa, IEa, IFa, Ib, IAb, IBb, IDb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, ID, IF, IG, Ia, IAa,IBa, IDa, IFa, IGa, Ib, IAb, IBb, IDb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from —CH₂OH and —CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH;and R₃; and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, ID, IE,IF, Ia, IAa, IBa, IDa, IEa, IFa, Ib, IAb, IBb, IDb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₃ is hydrogen or a —OR_(b) group for compounds of formula I, ID, IE,IF, IG, Ia, IDa, IEa, IFa, IGa, Ib, IDb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, ID, IF, IG, Ia, IAa,IBa, IDa, IFa, IGa, Ib, IAb, IBb, IDb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from —CH₂OH and —CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH;and R₁ is as defined as above.

Further preferred compounds include compounds of general formula I, IA,IB, ID, IE, IF, IG, Ia, IAa, IBa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb,IEb, IFb, and IGb, wherein:

Y is —O—; R₁ is —OH;

R₂ is a —C(═O)R_(a) group for compounds of formula I, IA, IB, ID, IE,IF, Ia, IAa, IBa, IDa, IEa, IFa, Ib, IAb, IBb, IDb, IEb, or IFb; and R₂is acetyl for compounds of formula IG, IGa or IGb; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₃ is hydrogen or a —OR_(b) group for compounds of formula I, ID, IE,IF, IG, Ia, IDa, IEa, IFa, IGa, Ib, IDb, IEb, IFb, or IGb; R₃ ishydrogen for compounds of formula IA, IAa, or IAb; and R₃ is a —OR_(b)group for compounds of formula IB, IBa or IBb; where R_(b) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(b)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. More preferred R₃are hydrogen and methoxy, being hydrogen the most preferred R₃ group;R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and—CH₂NHProt^(NH) for compounds of formula I, IA, IB, ID, IF, IG, Ia, IAa,IBa, IDa, IFa, IGa, Ib, IAb, IBb, IDb, IFb, or IGb; and R₄ is selectedfrom —CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula IE, IEa orIEb; where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.Particularly preferred R_(c) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from —CH₂OH and —CH₂NH₂. More preferably, R₄ may be—CH₂NH₂. Most preferred R₄ is —CH₂OH.

Further preferred compounds include compounds of general formula Ic,IAc, IBc, IDc, and IGc wherein:

R₁ is —OH;

and R₂; R₃; R_(a) and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula Ic,IAc, IBc, IDc, IFc, and IGc, wherein:

R₂ is a —C(═O)R_(a) group for compounds of formula Ic, IAc, IBc, IDc, orIFc; and R₂ is acetyl for compounds of formula IGc; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;and R₁; R₃; R_(b) are as defined as above.

Further preferred compounds include compounds of general formula Ic,IAc, IBc, IDc, IFc, and IGc, wherein:

R₃ is hydrogen or a —OR_(b) group for compounds of formula Ic, IDc, IFc,or IGc; R₃ is hydrogen for compounds of formula IAc; and R₃ is a —OR_(b)group for compounds of formula IBc; where R_(b) is a substituted orunsubstituted C₁-C₆ alkyl. Particularly preferred R_(b) is selected fromsubstituted or unsubstituted methyl, substituted or unsubstituted ethyl,substituted or unsubstituted n-propyl, substituted or unsubstitutedisopropyl, substituted or unsubstituted n-butyl, substituted orunsubstituted isobutyl, substituted or unsubstituted sec-butyl andsubstituted or unsubstituted tert-butyl. More preferred R₃ are hydrogenand methoxy, being hydrogen the most preferred R₃ group;and R₁; R₂; and R₃ are as defined as above.

Further preferred compounds include compounds of general formula Ic,IAc, IBc, IDc, IFc, and IGc, wherein:

R₁ is —OH;

R₃ is a —C(═O)R₃ group for compounds of formula Ic, IAc, IBc, IDc, orIFc; and R₃ is acetyl for compounds of formula IGc; where R₃ is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;and R₃; and R_(b) are as defined as above.

Further preferred compounds include compounds of general formula Ic,IAc, IBc, IDc, IFc, and IGc, wherein:

R₁ is —OH;

R₃ is hydrogen or a —OR_(b) group for compounds of formula Ic, IDc, IFc,or IGc; R₃ is hydrogen for compounds of formula IAc; and R₃ is a —OR_(b)group for compounds of formula IBc; where R_(b) is a substituted orunsubstituted C₁-C₆ alkyl. Particularly preferred R_(b) is selected fromsubstituted or unsubstituted methyl, substituted or unsubstituted ethyl,substituted or unsubstituted n-propyl, substituted or unsubstitutedisopropyl, substituted or unsubstituted n-butyl, substituted orunsubstituted isobutyl, substituted or unsubstituted sec-butyl andsubstituted or unsubstituted tert-butyl. More preferred R₃ are hydrogenand methoxy, being hydrogen the most preferred R₃ group;and R₂; and R_(a) are as defined as above.

Further preferred compounds include compounds of general formula Ic,IAc, IBc, IDc, IFc, and IGc, wherein:

R₂ is a —C(═O)R_(a) group for compounds of formula Ic, IAc, IBc, IDc, orIFc; and R₂ is acetyl for compounds of formula IGc; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₃ is hydrogen or a —OR_(b) group for compounds of formula Ic, IDc, IFc,or IGc; R₃ is hydrogen for compounds of formula IAc; and R₃ is a —OR_(b)group for compounds of formula IBc; where R_(b) is a substituted orunsubstituted C₁-C₆ alkyl. Particularly preferred R_(b) is selected fromsubstituted or unsubstituted methyl, substituted or unsubstituted ethyl,substituted or unsubstituted n-propyl, substituted or unsubstitutedisopropyl, substituted or unsubstituted n-butyl, substituted orunsubstituted isobutyl, substituted or unsubstituted sec-butyl andsubstituted or unsubstituted tert-butyl. More preferred R₃ are hydrogenand methoxy, being hydrogen the most preferred R₃ group;and R₁ is as defined as above.

Further preferred compounds include compounds of general formula Ic,IAc, IBc, IDc, IFc, and IGc, wherein:

R₁ is —OH;

R₂ is a —C(═O)R_(a) group for compounds of formula Ic, IAc, IBc, IDc, orIFc; and R₂ is acetyl for compounds of formula IGc; where R_(a) is asubstituted or unsubstituted C₁-C₆ alkyl. Particularly preferred R_(a)is selected from substituted or unsubstituted methyl, substituted orunsubstituted ethyl, substituted or unsubstituted n-propyl, substitutedor unsubstituted isopropyl, substituted or unsubstituted n-butyl,substituted or unsubstituted isobutyl, substituted or unsubstitutedsec-butyl and substituted or unsubstituted tert-butyl. Most preferred R₂is acetyl;R₃ is hydrogen or a —OR_(b) group for compounds of formula Ic, IDc, IFc,or IGc; R₃ is hydrogen for compounds of formula IAc; and R₃ is a —OR_(b)group for compounds of formula IBc; where R_(b) is a substituted orunsubstituted C₁-C₆ alkyl. Particularly preferred R_(b) is selected fromsubstituted or unsubstituted methyl, substituted or unsubstituted ethyl,substituted or unsubstituted n-propyl, substituted or unsubstitutedisopropyl, substituted or unsubstituted n-butyl, substituted orunsubstituted isobutyl, substituted or unsubstituted sec-butyl andsubstituted or unsubstituted tert-butyl. More preferred R₃ are hydrogenand methoxy, being hydrogen the most preferred R₃ group.

The following preferred substituents (where allowed by possiblesubstituent groups) apply to compounds of formula I, IA, IB, IC, ID, IE,IF, IG, Ia, IAa, IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb,IEb, IFb, IGb, Ic, IAc, IBc, IDc, IFc, and IGc: In compounds of thepresent invention, particularly preferred R₁ is —OH.

In compounds of the present invention, particularly preferred R₂ is a—C(═O)R_(a) group where R_(a) is a substituted or unsubstituted C₁-C₆alkyl. Particularly preferred R_(a) is selected from substituted orunsubstituted methyl, substituted or unsubstituted ethyl, substituted orunsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl and substituted orunsubstituted tert-butyl. Most preferred R₂ is acetyl.

In compounds of the present invention, particularly preferred R₃ ishydrogen or a —OR_(b) group where R_(b) is a substituted orunsubstituted C₁-C₆ alkyl. Particularly preferred R_(b) is selected fromsubstituted or unsubstituted methyl, substituted or unsubstituted ethyl,substituted or unsubstituted n-propyl, substituted or unsubstitutedisopropyl, substituted or unsubstituted n-butyl, substituted orunsubstituted isobutyl, substituted or unsubstituted sec-butyl andsubstituted or unsubstituted tert-butyl. More preferred R₃ are hydrogenand methoxy, being hydrogen the most preferred R₃ group.

In compounds of the present invention, particularly preferred R₄ isselected from H, —CH₂OH, —CH₂OC(═O)R_(c), —CH₂NH₂, and —CH₂NHProt^(NH)where R_(c) is a substituted or unsubstituted C₁-C₆ alkyl. Particularlypreferred R_(c) is selected from substituted or unsubstituted methyl,substituted or unsubstituted ethyl, substituted or unsubstitutedn-propyl, substituted or unsubstituted isopropyl, substituted orunsubstituted n-butyl, substituted or unsubstituted isobutyl,substituted or unsubstituted sec-butyl, and substituted or unsubstitutedtert-butyl. Most preferred R_(c) is methyl. More preferred R₄ isselected from H, CH₂OH and CH₂NH₂. Most preferred R₄ is —CH₂OH.

In compounds of general formula I, IA, IB, IC, ID, IE, IF, IG, Ia, IAa,IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb, and IGbparticularly preferred R₄ is selected from —CH₂OH, —CH₂OC(═O)R_(c),—CH₂NH₂, and —CH₂NHProt^(NH) for compounds of formula I, IA, IB, IC, ID,IF, IG, Ia, IAa, IBa, ICa, IDa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IFb,or IGb; and R₄ is selected from —CH₂NH₂, and —CH₂NHProt^(NH) forcompounds of formula IE, IEa or IEb; where R_(c) is a substituted orunsubstituted C₁-C₆ alkyl. Particularly preferred R_(c) is a substitutedor unsubstituted methyl, substituted or unsubstituted ethyl, substitutedor unsubstituted n-propyl, substituted or unsubstituted isopropyl,substituted or unsubstituted n-butyl, substituted or unsubstitutedisobutyl, substituted or unsubstituted sec-butyl, and substituted orunsubstituted tert-butyl. Most preferred R_(c) is methyl. More preferredR₄ is selected from CH₂OH and CH₂NH₂. Most preferred R₄ is —CH₂OH.

Being particularly preferred compounds of formula Ia, IAa, IBa, ICa,IDa, IFa, IGa when R₄ is —CH₂OH or —CH₂OC(═O)R_(c) and compounds offormula Ib, IAb, IBb, ICb, IDb, IEb, IFb, IGb when R₄ is —CH₂NH₂ or—CH₂NHProt^(NH).

In compounds of the present invention, particularly preferred Y is —NH—.

Alternatively, in compounds of the present invention, particularlypreferred Y is —O—.

Preferred compounds according to the present invention include:

-   -   Compounds of formula I, IA, IB, IC, ID, IF, IG, Ia, IAa, IBa,        ICa, IDa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IFb, and IGb        wherein:        -   R₄ is selected from —CH₂OH and —CH₂OC(═O)R_(c);        -   Being particularly preferred compounds of formula Ia, IAa,            IBa, ICa, IDa, IFa, and IGa and/or compounds where FU is            —CH₂OH.    -   Compounds of formula I, IA, IB, IC, ID, IE IF, IG, Ia, IAa, IBa,        ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb, and        IGb wherein        -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH); and        -   Prot^(NH) is a protecting group for amino.        -   Being particularly preferred compounds of formula Ib, IAb,            IBb, ICb, IDb, IEb, IFb, and IGb and/or compounds where FU            is —CH₂NH₂.    -   Compounds of formula Ic, IAc, IBc, IDc, IFc, IGc wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula Ic, IAc,            IBc, IDc, or IFc; and R₂ is acetyl for compounds of formula            IGc;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            Ic, IDc, IFc, IGc; R₃ is hydrogen for compounds of formula            IAc; or R₃ is a —OR_(b) group for compounds of formula IBc;        -   R_(a) is selected from hydrogen, and substituted or            unsubstituted C₁-C₆ alkyl; and        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl.

Particularly preferred compounds according to the present inventioninclude:

-   -   Compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa, IBa, ICa,        IFa, IGa, Ib, IAb, IBb, ICb, IFb, and IGb wherein        -   Y is —NH—;        -   R₄ is selected from —CH₂OH, and —CH₂OC(═O)R_(c);        -   and        -   R_(c) is selected from substituted or unsubstituted C₁-C₁₂            alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and            substituted or unsubstituted C₂-C₁₂ alkynyl.        -   Being more preferred compounds of formula Ia, IAa, IBa, ICa,            IFa, IGa and/or compounds where R₄ is —CH₂OH.    -   Compounds of formula I, IA, IB, ID, IF, IG, Ia, IAa, IBa, IDa,        IFa, IGa, Ib, IAb, IBb, IDb, IFb, and IGb wherein        -   Y is —O—;        -   R₄ is selected from —CH₂OH and —CH₂OC(═O)R_(c);        -   and        -   R_(c) is selected from substituted or unsubstituted C₁-C₁₂            alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, and            substituted or unsubstituted C₂-C₁₂ alkynyl.        -   Being more preferred compounds of formula Ia, IAa, IBa, IDa,            IFa, IGa and/or compounds where R₄ is —CH₂OH.    -   Compounds of formula I, IA, IB, IC, IE, IF, IG, Ia, IAa, IBa,        ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IEb, IFb, and IGb wherein        -   Y is —NH—;        -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH);        -   and        -   Prot^(NH) is a protecting group for amino.        -   Being more preferred compounds of formula Ib, IAb, IBb, ICb,            IEb, IFb, IGb and/or compounds where R₄ is —CH₂NH₂.    -   Compounds of formula I, IA, IB, ID, IE, IF, IG, Ia, IAa, IBa,        IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb, IEb, IFb, and IGb wherein        -   Y is —O—;        -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH);        -   and        -   Prot^(NH) is a protecting group for amino.        -   Being more preferred compounds of formula Ib, IAb, IBb, IDb,            IEb, IFb, IGb and/or compounds where FU is —CH₂NH₂.    -   Compounds of formula I, IA, IB, IC, ID, IF, IG, Ia, IAa, IBa,        ICa, IDa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IFb, IGb wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, ID, IF, Ia, IAa, IBa, ICa, IDa, IFa, Ib, IAb, IBb,            ICb, IDb, or IFb; and R₂ is acetyl for compounds of formula            IG, IGa or IGb;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            I, IC, ID, IF, IG, Ia, ICa, IDa, IFa, IGa, Ib, ICb, IDb,            IFb, or IGb; R₃ is hydrogen for compounds of formula IA, IAa            or IAb; or R₃ is a —OR_(b) group for compounds of formula            IB, IBa or IBb;        -   R₄ is selected from —CH₂OH, and —CH₂OC(═O)R_(c);        -   R_(a) is selected from hydrogen, and substituted or            unsubstituted C₁-C₆ alkyl;        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl; and        -   R_(c) is substituted or unsubstituted C₁-C₆ alkyl.        -   Being more preferred compounds of formula Ia, IAa, IBa, ICa,            IDa, IFa, IGa and/or compounds where R₄ is —CH₂OH.    -   Compounds of formula I, IA, IB, IC, ID, IE, IF, IG, Ia, IAa,        IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb,        and IGb wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, ID, IF, Ia, IAa, IBa, ICa, IDa, IFa, Ib, IAb, IBb,            ICb, IDb, or IFb; and R₂ is acetyl for compounds of formula            IG, IGa or IGb;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            I, IC, ID, IE, IF, IG, Ia, ICa, IDa, IEa, IFa, IGa, Ib, ICb,            IDb, IEb, IFb, or IGb; R₃ is hydrogen for compounds of            formula IA, IAa or IAb; or R₃ is a —OR_(b) group for            compounds of formula IB, IBa or IBb;        -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH);        -   R_(a) is selected from hydrogen, and substituted or            unsubstituted C₁-C₆ alkyl;        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl; and        -   Prot^(NH) is a protecting group for amino.        -   Being more preferred compounds of formula Ib, IAb, IBb, ICb,            IDb, IEb, IFb, IGb and/or compounds where FU is —CH₂NH₂.    -   Compounds of formula Ic, IAc, IBc, IDc, IFc, IGc wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula Ic, IAc,            IBc, IDc, or IFc; and R₂ is acetyl for compounds of formula            IGc;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            Ic, IDc, IFc, IGc; R₃ is hydrogen for compounds of formula            IAc; or R₃ is a —OR_(b) group for compounds of formula IBc;        -   R_(a) is substituted or unsubstituted C₁-C₆ alkyl; and        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl.

More preferred compounds according to the present invention include

-   -   Compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa, IBa, ICa,        IFa, IGa, Ib, IAb, IBb, ICb, IFb, and IGb wherein        -   Y is —NH—;        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, IF, Ia, IAa, IBa, ICa, IFa, Ib, IAb, IBb, ICb, or            IFb; and R₂ is acetyl for compounds of formula IG, IGa or            IGb;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            I, IC, IF, IG, Ia, ICa, IFa, IGa, Ib, ICb, IFb, or IGb; R₃            is hydrogen for compounds of formula IA, IAa or IAb; or R₃            is a —OR_(b) group for compounds of formula IB, IBa or IBb;        -   R₄ is —CH₂OH;        -   R_(a) is selected from hydrogen and substituted or            unsubstituted C₁-C₆ alkyl; and        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl.        -   Being particularly more preferred compounds of formula Ia,            IAa, or IBa, ICa, IFa, IGa.    -   Compounds of formula I, IA, IB, ID, IF, IG, Ia, IAa, IBa, IDa,        IFa, IGa, Ib, IAb, IBb, IDb, IFb, and IGb wherein        -   Y is —O—;        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, ID, IF, Ia, IAa, IBa, IDa, IFa, Ib, IAb, IBb, IDb, or            IFb; and R₂ is acetyl for compounds of formula IG, IGa or            IGb;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            I, ID, IF, IG, Ia, IDa, IFa, IGa, Ib, IDb, IFb, or IGb; R₃            is hydrogen for compounds of formula IA, IAa or IAb; or R₃            is a —OR_(b) group for compounds of formula IB, IBa or IBb;        -   R₄ is —CH₂OH;        -   R_(a) is selected from hydrogen and substituted or            unsubstituted C₁-C₆ alkyl; and        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl.        -   Being particularly more preferred compounds of formula Ia,            IAa, IBa, IDa, IFa, or IGa.    -   Compounds of formula I, IA, IB, IC, IE, IF, IG, Ia, IAa, IBa,        ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IEb, IFb, and IGb wherein        -   Y is —NH—;        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, IE, IF, Ia, IAa, IBa, ICa, IEa, IFa, Ib, IAb, IBb,            ICb, IEb or IFb; and R₂ is acetyl for compounds of formula            IG, IGa or IGb;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            I, IC, IE, IF, IG, Ia, ICa, IEa, IFa, IGa, Ib, ICb, IEb,            IFb, or IGb; R₃ is hydrogen for compounds of formula IA, IAa            or IAb; or R₃ is a —OR_(b) group for compounds of formula            IB, IBa or IBb;        -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH);        -   R_(a) is selected from hydrogen and substituted or            unsubstituted C₁-C₆ alkyl;        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl; and        -   Prot^(NH) is a protecting group for amino.        -   Being particularly more preferred compounds of formula Ib,            IAb, IBb, ICb, IEb, IFb, IGb and/or compounds where R₄ is            —CH₂NH₂.    -   Compounds of formula I, IA, IB, ID, IE, IF, IG, Ia, IAa, IBa,        IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb, IEb, IFb, and IGb wherein        -   Y is —O—;        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, ID, IE, IF, Ia, IAa, IBa, IDa, IEa, IFa, Ib, IAb, IBb,            IDb, IEb or IFb; and R₂ is acetyl for compounds of formula            IG, IGa or IGb;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            I, ID, IE, IF, IG, Ia, IDa, IEa, IFa, IGa, Ib, IDb, IEb,            IFb, or IGb; R₃ is hydrogen for compounds of formula IA, IAa            or IAb; or R₃ is a —OR_(b) group for compounds of formula            IB, IBa or IBb;        -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH);        -   R_(a) is selected from hydrogen and substituted or            unsubstituted C₁-C₆ alkyl;        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl; and        -   Prot^(NH) is a protecting group for amino.        -   Being particularly more preferred compounds of formula Ib,            IAb, IBb, IDb, IEb, IFb, IGb and/or compounds where R₄ is            CH₂NH₂.    -   Compounds of formula I, IA, IB, IC, ID, IF, IG, Ia, IAa, IBa,        ICa, IDa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IFb, and IGb wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, ID, IF, Ia, IAa, IBa, ICa, IDa, IFa, Ib, IAb, IBb,            ICb, IDb or IFb; and R₂ is acetyl for compounds of formula            IG, IGa or IGb;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            I, IC, ID, IF, IG, Ia, ICa, IDa, IFa, IGa, Ib, ICb, IDb,            IFb, or IGb; R₃ is hydrogen for compounds of formula IA, IAa            or IAb; or R₃ is a —OR_(b) group for compounds of formula            IB, IBa or IBb;        -   R₄ is —CH₂OH;        -   R_(a) is substituted or unsubstituted C₁-C₆ alkyl; and        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl.        -   Being particularly more preferred compounds of formula Ia,            IAa, IBa, ICa, IDa, IFa, or IGa.    -   Compounds of formula I, IA, IB, IC, ID, IE, IF, IG, Ia, IAa,        IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb,        and IGb wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, ID, IE, IF, Ia, IAa, IBa, ICa, IDa, IEa, IFa, Ib,            IAb, IBb, ICb, IDb, IEb or IFb; and R₂ is acetyl for            compounds of formula IG, IGa or IGb;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            I, IC, ID, IE, IF, IG, Ia, ICa, IDa, IEa, IFa, IGa, Ib, ICb,            IDb, IEb, IFb, or IGb; R₃ is hydrogen for compounds of            formula IA, IAa or IAb; or R₃ is a —OR_(b) group for            compounds of formula IB, IBa or IBb;        -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH);        -   R_(a) is substituted or unsubstituted C₁-C₆ alkyl;        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl; and        -   Prot^(NH) is a protecting group for amino.        -   Being particularly more preferred compounds of formula Ib,            IAb, IBb, ICb, IDb, IEb, IFb, IGb and/or compounds where R₄            is —CH₂NH₂.    -   Compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa, IBa, ICa,        IFa, IGa, Ib, IAb, IBb, ICb, IFb, and IGb wherein        -   Y is —NH—;        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, IF, Ia, IAa, IBa, ICa, IFa, Ib, IAb, IBb, ICb, or            IFb; and R₂ is acetyl for compounds of formula IG, IGa or            IGb;        -   R₃ is hydrogen or a —OR_(b) group for compounds of formula            I, IC, IF, IG, Ia, ICa, IFa, IGa, Ib, ICb, IFb, or IGb; R₃            is hydrogen for compounds of formula IA, IAa or IAb; or R₃            is a —OR_(b) group for compounds of formula IB, IBa or IBb;        -   R₄ is —CH₂OC(═O)RC;        -   R_(a) is selected from hydrogen and substituted or            unsubstituted C₁-C₆ alkyl;        -   R_(b) is substituted or unsubstituted C₁-C₆ alkyl; and        -   R_(c) is a substituted or unsubstituted C₁-C₆ alkyl.        -   Being more preferred compounds of formula Ia, IAa, IBa, ICa,            IFa, or IGa.    -   Compounds of formula Ic, IAc, IBc, IDc, IFc, and IGc wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula Ic, IAc,            IBc, IDc, or IFc; and R₂ is acetyl for compounds of formula            IGc;        -   R₃ is hydrogen or methoxy for compounds of formula Ic, IDc,            IFc, or IGc; R₃ is hydrogen for compounds of formula IAc; or            R₃ is methoxy for compounds of formula IBc; and        -   R_(a) is substituted or unsubstituted C₁-C₆ alkyl.

Particularly more preferred compounds according to the present inventioninclude:

-   -   Compounds of formula I, IA, IB, IC, IF, IG, Ia, IAa, IBa, ICa,        IFa, IGa, Ib, IAb, IBb, ICb, IFb, and IGb wherein        -   Y is —NH—;        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, IF, Ia, IAa, IBa, ICa, IFa, Ib, IAb, IBb, ICb, or            IFb; and R₂ is acetyl for compounds of formula IG, IGa or            IGb;        -   R₃ is hydrogen or methoxy for compounds of formula I, IC,            IF, IG, Ia, ICa, IFa, IGa, Ib, ICb, IFb, or IGb; R₃ is            hydrogen for compounds of formula IA, IAa or IAb; and        -   R₃ is methoxy for compounds of formula IB, IBa or IBb;        -   R₄ is —CH₂OH; and        -   R_(a) is substituted or unsubstituted C₁-C₆ alkyl.        -   Being even more preferred compounds of formula Ia, IAa, IBa,            ICa, IFa, IGa.    -   Compounds of formula I, IA, IB, ID, IF, IG, Ia, IAa, IBa, IDa,        IFa, IGa, Ib, IAb, IBb, IDb, IFb, and IGb wherein        -   Y is —O—;        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, ID, IF, Ia, IAa, IBa, IDa, IFa, Ib, IAb, IBb, IDb, or            IFb; and R₂ is acetyl for compounds of formula IG, IGa or            IGb;        -   R₃ is hydrogen or methoxy for compounds of formula I, ID,            IF, IG, Ia, IDa, IFa, IGa, Ib, IDb, IFb, or IGb; R₃ is            hydrogen for compounds of formula IA, IAa or IAb; or R₃ is            methoxy for compounds of formula IB, IBa or IBb;        -   R₄ is —CH₂OH; and        -   R_(a) is substituted or unsubstituted C₁-C₆ alkyl.        -   Being even more preferred compounds of formula Ia, IAa, IBa,            IDa, IEa, IFa, IGa.    -   Compounds of formula I, IA, IB, IC, IE, IF, IG, Ia, IAa, IBa,        ICa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IEb, IFb, and IGb wherein        -   Y is —NH—;        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, IE, IF, Ia, IAa, IBa, ICa, IEa, IFa, Ib, IAb, IBb,            ICb, IEb or IFb; and R₂ is acetyl for compounds of formula            IG, IGa or IGb;        -   R₃ is hydrogen or methoxy for compounds of formula I, IC,            IE, IF, IG, Ia, ICa, IEa, IFa, IGa, Ib, ICb, IEb, IFb, or            IGb; R₃ is hydrogen for compounds of formula IA, IAa or IAb;            or R₃ is methoxy for compounds of formula IB, IBa or IBb;        -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH);        -   R_(a) is substituted or unsubstituted C₁-C₆ alkyl; and        -   Prot^(NH) is a protecting group for amino.        -   Being even more preferred compounds of formula Ib, IAb, IBb,            ICb, IEb, IFb, IGb and/or compounds where R₄ is —CH₂NH₂.    -   Compounds of formula I, IA, IB, ID, IE, IF, IG, Ia, IAa, IBa,        IDa, IEa, IFa, IGa, Ib, IAb, IBb, IDb, IEb, IFb, and IGb wherein        -   Y is —O—;        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, ID, IE, IF, Ia, IAa, IBa, IDa, IEa, IFa, Ib, IAb, IBb,            IDb, IEb or IFb; and R₂ is acetyl for compounds of formula            IG, IGa or IGb;        -   R₃ is hydrogen or methoxy for compounds of formula I, ID,            IE, IF, IG, Ia, IDa, IEa, IFa, IGa, Ib, IDb, IEb, IFb, or            IGb; R₃ is hydrogen for compounds of formula IA, IAa or IAb;            or R₃ is methoxy for compounds of formula IB, IBa or IBb;        -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH);        -   R_(a) is substituted or unsubstituted C₁-C₆ alkyl; and        -   Prot^(NH) is a protecting group for amino.        -   Being even more preferred compounds of formula Ib, IAb, IBb,            IDb, IEb, IFb, IGb and/or compounds where R₄ is —CH₂NH₂.    -   Compounds of formula I, IA, IB, IC, ID, IF, IG, Ia, IAa, IBa,        ICa, IDa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IFb, and IGb wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, ID, IF, Ia, IAa, IBa, ICa, IDa, IFa, Ib, IAb, IBb,            ICb, IDb, or IFb; and R₂ is acetyl for compounds of formula            IG, IGa or IGb;        -   R₃ is hydrogen or methoxy for compounds of formula I, IC,            ID, IF, IG, Ia, ICa, IDa, IFa, IGa, Ib, ICb, IDb, IFb, and            IGb; R₃ is hydrogen for compounds of formula IA, IAa or IAb;            or R₃ is methoxy for compounds of formula IB, IBa or IBb;        -   R₄ is —CH₂OH; and        -   R_(a) is selected from methyl, ethyl, n-propyl, isopropyl            and butyl, including n-butyl, sec-butyl, isobutyl and            tert-butyl.        -   Being even more preferred compounds of formula Ia, IAa, IBa,            ICa, IDa, IEa, IFa, or IGa.    -   Compounds of formula I, IA, IB, IC, ID, IE, IF, IG, Ia, IAa,        IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb,        and IGb wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula I, IA,            IB, IC, ID, IE, IF, Ia, IAa, IBa, ICa, IDa, IEa, IFa, Ib,            IAb, IBb, ICb, IDb, IEb or IFb; and R₂ is acetyl for            compounds of formula IG, IGa or IGb;        -   R₃ is hydrogen or a methoxy for compounds of formula I, IC,            ID, IE, IF, IG, Ia, ICa, IDa, IEa, IFa, IGa, Ib, ICb, IDb,            IEb, IFb, and IGb; R₃ is hydrogen for compounds of formula            IA, IAa or IAb; or R₃ is methoxy for compounds of formula            IB, IBa or IBb;        -   R₄ is selected from —CH₂NH₂ and —CH₂NHProt^(NH);        -   R_(a) is selected from methyl, ethyl, n-propyl, isopropyl            and butyl, including n-butyl, sec-butyl, isobutyl and            tert-butyl; and        -   Prot^(NH) is a protecting group for amino.        -   Being even more preferred compounds of formula Ib, IAb, IBb,            ICb, IDb, IEb, IFb, IGb and/or compounds where R₄ is            —CH₂NH₂.    -   Compounds of formula Ic or IAc, IDc, IFc, and IGc wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula Ic, IAc,            IDc, or IFc; and R₂ is acetyl for compounds of formula IGc;        -   R₃ is hydrogen; and        -   R_(a) is selected from methyl, ethyl, n-propyl, isopropyl            and butyl, including n-butyl, sec-butyl, isobutyl and            tert-butyl.    -   Compounds of formula Ic, IBc, IDc, IFc, and IGc wherein        -   R₂ is a —C(═O)R_(a) group for compounds of formula Ic, IBc,            IDc, or IFc; and R₂ is acetyl for compounds of formula IGc;        -   R₃ is methoxy; and        -   R_(a) is selected from methyl, ethyl, n-propyl, isopropyl            and butyl, including n-butyl, sec-butyl, isobutyl and            tert-butyl.

Even more preferred compounds according to the present inventioninclude:

-   -   Compounds of formula I, IA, IC, IF, IG, Ia, IAa, ICa, IFa, IGa,        Ib, IAb, ICb, IFb, and IGb wherein        -   Y is —NH—;        -   R₂ is acetyl;        -   R₃ is hydrogen; and        -   R₄ is —CH₂OH.        -   Being most preferred compounds of formula Ia, IAa, ICa, IFa,            or IGa.    -   Compounds of formula I, IA, ID, IF, IG, Ia, IAa, IDa, IFa, IGa,        Ib, IAb, IDb, IFb, and IGb wherein        -   Y is —O—;        -   R₂ is acetyl;        -   R₃ is hydrogen; and        -   R₄ is —CH₂OH.        -   Being most preferred compounds of formula Ia, IAa, IDa, IFa,            or IGa    -   Compounds of formula I, IA, IC, IE, IF, IG, Ia, IAa, ICa, IEa,        IFa, IGa, Ib, IAb, ICb, IEb, IFb, and IGb wherein        -   Y is —NH—;        -   R₂ is acetyl;        -   R₃ is hydrogen; and        -   R₄ is —CH₂NH₂.

Being most preferred compounds of formula Ib, IAb, ICb, IEb, IFb, orIGb.

-   -   Compounds of formula I, IA, ID, IE, IF, IG, Ia, IAa, IDa, IEa,        IFa, IGa, Ib, IAb, IDb, IEb, IFb, and IGb wherein        -   Y is —O—;        -   R₂ is acetyl;        -   R₃ is hydrogen; and        -   R₄ is —CH₂NH₂.        -   Being most preferred compounds of formula Ib, IAb, IDb, IEb,            IFb, or IGb.    -   Compounds of formula I, IA, IC, ID, IF, IG, Ia, IAa, ICa, IDa,        IFa, IGa, Ib, IAb, ICb, IDb, IFb, and IGb wherein        -   R₂ is acetyl;        -   R₃ is hydrogen; and        -   R₄ is —CH₂OH.        -   Being most preferred compounds of formula Ia, IAa, ICa, IDa,            IFa or IGa.    -   Compounds of formula I, IA, IC, ID, IF, IG, Ia, IAa, ICa, IDa,        IFa, IGa, Ib, IAb, ICb, IDb, IFb, and IGb wherein        -   R₁ is —OH;        -   R₂ is acetyl;        -   R₃ is hydrogen; and        -   R₄ is —CH₂OH.        -   Being most preferred compounds of formula Ia, IAa, ICa, IDa,            IFa or IGa.    -   Compounds of formula I, IA, IC, ID, IE, IF, IG, Ia, IAa, ICa,        IDa, IEa, IFa, IGa, Ib, IAb, ICb, IDb, IEb, IFb, and IGb wherein        -   R₂ is acetyl;        -   R₃ is hydrogen; and        -   R₄ is —CH₂NH₂.

Being most preferred compounds of formula Ib, IAb, ICb, IDb, IEb, IFb,or IGb.

-   -   Compounds of formula Ic or IAc, IDc, IFc, IGc wherein        -   R₂ is acetyl; and        -   R₃ is hydrogen.    -   Compounds of formula Ic or IBc, IDc, IFc, IGc wherein        -   R₂ is acetyl; and        -   R₃ is methoxy.    -   A compound according to the present invention of formula:

or a pharmaceutically acceptable salt or ester thereof.

Being particularly preferred a compound of formula:

or a pharmaceutically acceptable salt or ester thereof.

-   -   A compound according to the present invention of formula:

or a pharmaceutically acceptable salt or ester thereof.

Being particularly preferred a compound of formula:

or a pharmaceutically acceptable salt or ester thereof.

Being more preferred a compound of formula:

or a pharmaceutically acceptable salt or ester thereof.

Being even more preferred compounds according to the present inventionare compounds of formula:

or a pharmaceutically acceptable salt or ester thereof.Further preferred compounds according to the present invention arecompounds of formula:

or a pharmaceutically acceptable salt or ester thereof.

In a further embodiment, according to the present invention preferredcompounds are compounds of formula:

or a pharmaceutically acceptable salt or ester thereof.

Further preferred compounds include a compound of formula:

or a pharmaceutically acceptable salt or ester thereof.

Further preferred compounds include a compound of formula:

or a pharmaceutically acceptable salt or ester thereof.

In additional preferred embodiments, the preferences described above forthe different substituents are combined. The present invention is alsodirected to such combinations of preferred substitutions (where allowedby possible substituent groups) in compounds of formula I, IA, IB, IC,ID, IE, IF, IG, Ia, IAa, IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb,ICb, IDb, IEb, IFb, IGb, Ic, IAc, IBc, IDc, IFc or IGc according to thepresent invention.

For the avoidance of doubt, the compounds above may be the drug moiety Dand are covalently attached via a hydroxy or amine group to (X)_(b) ifany, or (AA)_(w) if any, or to (T)_(g) if any, or (L). Thus, whenconjugated, a covalent bond replaces a proton on a hydroxy or aminegroup on the compound.Preferred drug conjugates according to the the present invention aregiven below. The preferred definitions of (X)_(b), (AA)_(w), (T)_(g),and (L) as set out below are applicable to all of the drug moiety Dcompounds described above. Preferred drug conjugates according to thethe present invention include:

-   -   a drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention wherein L is a linker group selected from the        group consisting of:

-   -   -   wherein        -   the wavy lines indicate the point of covalent attachments to            an Ab (the wavy line to the right) and to (T)_(g) if any, or            (AA)_(w) if any, or to (X)_(b) if any, or to D (the wavy            line to the left);        -   R₁₉ is selected from —C₁-C₁₂ alkylene-, —C₃-C₈ carbocyclo,            —O—(C₁-C₁₂ alkylene), —C₆-C₁₈ arylene in one or more rings            which may optionally be substituted with one or more            substituents R_(x), —C₁-C₁₂ alkylene-C₆-C₁₈ arylene- wherein            the arylene group is in one or more rings which may            optionally be substituted with one or more substituents            R_(x), —C₆-C₁₈ arylene-C₁-C₁₂ alkylene- wherein the arylene            group is in one or more rings which may optionally be            substituted with one or more substituents R_(x), —C₁-C₁₂            alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈ carbocyclo)-C₁-C₁₂            alkylene-, —C₅-C₁₄ heterocyclo- wherein said heterocyclo            group may be a saturated or unsaturated group having one or            more rings and comprising at least one oxygen, nitrogen or            sulphur atom in said ring(s), said group optionally being            substituted with one or more substituents R_(x), —C₁-C₁₂            alkylene-(C₅-C₁₄ heterocyclo)- wherein said heterocyclo            group may be a saturated or unsaturated group having one or            more rings and comprising at least one oxygen, nitrogen or            sulphur atom in said ring(s), said group optionally being            substituted with one or more substituents R_(x), —(C₅-C₁₄            heterocyclo)-C₁-C₁₂ alkylene- wherein said heterocyclo group            may be a saturated or unsaturated group having one or more            rings and comprising at least one oxygen, nitrogen or            sulphur atom in said ring(s), said group optionally being            substituted with one or more substituents R_(x),            —(OCH₂CH₂)_(r), and —CH₂—(OCH₂CH₂)_(r), wherein each of the            above alkylene substituents whether alone or attached to            another moiety the carbon chain may optionally be            substituted by one or more substituents R_(x);        -   R₃₀ is a —C₁-C₆ alkylene- group;        -   M is selected from the group consisting of —C₁-C₆ alkylene-,            —C₁-C₆ alkylene-(C₃-C₈ carbocyclo)-, —(CH₂CH₂O)_(s)—, —C₁-C₆            alkylene-(C₃-C₈ carbocyclo)-CON(H or C₁-C₆ alkyl)-C₁-C₆            alkylene-, phenylene which may optionally be substituted            with one or more substituents R_(x), phenylene-C₁-C₆            alkylene- wherein the phenylene moiety may optionally be            substituted with one or more substituents R_(x) and —C₁-C₆            alkylene-CON(H or C₁-C₆alkyl)C₁-C₆ alkylene-;        -   Q is selected from the group consisting of —N(H or C₁-C₆            alkyl)phenylene- and —N(H or C₁-C₆alkyl)-(CH₂)_(s);        -   r is an integer ranging from 1 to 10; and        -   s is an integer ranging from 1 to 10.

    -   a drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention wherein L is selected from the group        consisting of:

-   -   -   wherein:        -   the wavy lines indicate the point of covalent attachments to            an Ab (the wavy line to the right) and to (T)_(g) if any, or            (AA)_(w) if any, or to (X)_(b) if any, or to D (the wavy            line to the left);        -   R₁₉ is selected from —C₁-C₁₂ alkylene-, —O—(C₁-C₁₂            alkylene), —C₆-C₁₂ arylene in one or more rings which may            optionally be substituted with one or more substituents            R_(x), —C₁-C₁₂ alkylene-C₆-C₁₂ arylene- wherein the arylene            group is in one or more rings which may optionally be            substituted with one or more substituents R_(x), —C₆-C₁₂            arylene-C₁-C₁₂ alkylene- wherein the arylene group is in one            or more rings which may optionally be substituted with one            or more substituents R_(x), —C₅-C₁₂ heterocyclo- wherein            said heterocyclo group may be a saturated or unsaturated            group having one or more rings and comprising at least one            oxygen, nitrogen or sulphur atom in said ring(s), said group            optionally being substituted with one or more substituents            R_(x), —C₁-C₁₂ alkylene-(C₅-C₁₂ heterocyclo)- wherein said            heterocyclo group may be a saturated or unsaturated group            having one or more rings and comprising at least one oxygen,            nitrogen or sulphur atom in said ring(s), said group            optionally being substituted with one or more substituents            R_(x), —(C₅-C₁₂ heterocyclo)-C₁-C₁₂ alkylene- wherein said            heterocyclo group may be a saturated or unsaturated group            having one or more rings and comprising at least one oxygen,            nitrogen or sulphur atom in said ring(s), said group            optionally being substituted with one or more substituents            R_(x), —(OCH₂CH₂)_(r)-, and —CH₂—(OCH₂CH₂)_(r)— wherein each            of the above alkylene substituents whether alone or attached            to another moiety the carbon chain may optionally be            substituted by one or more substituents R_(x);        -   R₃₀ is a —C₁-C₆ alkylene- group;        -   M is selected from the group consisting of —C₁-C₆ alkylene-,            —C₁-C₆ alkylene-(C₃-C₈ carbocyclo)- and phenylene which may            optionally be substituted with one or more substituents            R_(x); and        -   r is an integer ranging from 1-6.

    -   a drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention selected from formulas (IV), (V) and (VI):

-   -   -   wherein:        -   X and T are extending groups as defined herein;        -   each AA is independently an amino acid unit as defined            herein;        -   w is an integer ranging from 0 to 12;        -   b is an integer of 0 or 1;        -   g is an integer of 0 or 1;        -   where b+g+w is optionally not 0;        -   D is a drug moiety; Ab is a moiety comprising at least one            antigen binding site;        -   n is the ratio of the group            [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein L is as defined in            formula (IV), (V) or (VI) to the moiety comprising at least            one antigen binding site and is in the range from 1 to 20;        -   R₁₉ is selected from —C₁-C₈ alkylene-, —O—(C₁-C₈ alkylene),            —C₁-C₈ alkylene-C₆-C₁₂ arylene- wherein the arylene group is            in one or more rings which may optionally be substituted            with one or more substituents R_(x), and —C₆-C₁₂            arylene-C₁-C₈ alkylene-wherein the arylene group is in one            or more rings which may optionally be substituted with one            or more substituents R_(x), wherein each of the above            alkylene substituents whether alone or attached to another            moiety the carbon chain may optionally be substituted by one            or more substituents R_(x);        -   R₃₀ is a —C₂-C₄ alkylene- group; and        -   M is selected from the group consisting of —C₁-C₃ alkylene-            and —C₁-C₃ alkylene-(C₅-C₇ carbocyclo)-.

    -   a drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention, selected from formulas (IV), (V) and (VI):

-   -   -   wherein:        -   X and T are extending groups that may be the same or            different;        -   each AA is independently an amino acid unit;        -   w is an integer ranging from 0 to 12;        -   b is an integer of 0 or 1;        -   g is an integer of 0 or 1;        -   where b+g+w is optionally not 0;        -   D is a drug moiety;        -   Ab is a moiety comprising at least one antigen binding site;        -   n is the ratio of the group            [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein L is as defined in            formulas (IV), (V) or (VI) to the moiety comprising at least            one antigen binding site and is in the range from 1 to 20;        -   R₁₉ is selected from —C₁-C₆ alkylene-, phenylene-C₁-C₆            alkylene- wherein the phenylene group may optionally be            substituted with one or more substituents R_(x) selected            from the group consisting of alkyl groups having from 1 to 6            carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,            halogen atoms, nitro groups and cyano groups, wherein each            of the above alkylene substituents whether alone or attached            to another moiety in the carbon chain may optionally be            substituted by one or more substituents R_(x) selected from            the group consisting of alkyl groups having from 1 to 6            carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,            aryl groups having from 6 to 12 carbon atoms, halogen atoms,            nitro groups and cyano groups, and preferably R₁₉ is a            —C₁-C₆ alkylene group;        -   R₃₀ is a —C₂-C₄ alkylene- group; and        -   M is —C₁-C₃ alkylene-(C₅-C₇carbocyclo)-.

    -   It is preferred that in the definition of the drug conjugate of        formula [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab, L is as        defined in the preferred definitions for said group above and        (AA)_(w) is of formula (II):

-   -   -   wherein the wavy lines indicate the point of covalent            attachments to (X)_(b) if any, or to the drug moiety (the            wavy line to the left) and to (T)_(g) if any, or to the            linker (the wavy line to the right); and        -   R₂₁ is, at each occurrence, selected from the group            consisting of hydrogen, methyl, isopropyl, isobutyl,            sec-butyl, benzyl, p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃,            —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH,            —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂, —(CH₂)₃NHCOCH₃,            —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,            —(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂,            —(CH₂)₄NHCONH₂, —CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-,            3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

-   -   -   and w is an integer ranging from 0 to 12.

    -   a drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the first        aspect of the present invention, wherein L is as defined in the        preferred definitions for said group above and (AA)_(w) is of        formula (II) wherein:        -   R₂₁ is selected, at each occurrence, from the group            consisting of hydrogen, methyl, isopropyl, sec-butyl,            benzyl, indolylmethyl, —(CH₂)₃NHCONH₂, —(CH₂)₄NH₂,            —(CH₂)₃NHC(═NH)NH₂ and —(CH₂)₄NHC(═NH)NH₂; and        -   w is an integer ranging from 0 to 6.

    -   a drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the first        aspect of the present invention, wherein L is as defined in the        preferred definitions for said group above, wherein w is 0 or 2,        and when w is 2, then (AA)_(w) is of formula (III) wherein:

-   -   -   the wavy lines indicate the point of covalent attachments to            (X)_(b) if any, or to the drug moiety (the wavy line to the            left) and to (T)_(g) if any, or to the linker (the wavy line            to the right);        -   R₂₂ is selected from methyl, benzyl, isopropyl, sec-butyl            and indolylmethyl; and        -   R₂₃ is selected from methyl, —(CH₂)₄NH₂, —(CH₂)₃NHCONH₂ and            —(CH₂)₃NHC(═NH)NH₂.

    -   In embodiments of the present invention b+g+w is not 0. In        further embodiments, b+w is not 0. In yet further embodiments,        when w is not 0, then b is 1. Further, it is preferred that in        the definition of the drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab, L and (AA)_(w) are as        defined in the preferred definitions for said groups above and X        is an extending group selected from:        -   where D is conjugated via an amine group (for example where            Z is —NH—):            -   —COO—(C₁-C₆ alkylene)NH—;            -   —COO—CH₂-(phenylene which may optionally be substituted                with one or more substituents R_(x))—NH—;            -   —COO—(C₁-C₆ alkylene)NH—COO—CH₂-(phenylene which may                optionally be substituted with one or more substituents                R_(x))—NH—;            -   —COCH₂NH—COCH₂—NH—;            -   —COCH₂NH—;            -   —COO—(C₁-C₆ alkylene)S—;            -   —COO—(C₁-C₆ alkylene)NHCO(C₁-C₆ alkylene)S—; or        -   where D is conjugated via an hydroxy group (for example            where Z is —O—):            -   —CONH—(C₁-C₆ alkylene)NH—;            -   —COO—CH₂-(phenylene which may optionally be substituted                with one or more substituents R_(x))—NH—;            -   —CONH—(C₁-C₆ alkylene)NH—COO—CH₂-(phenylene which may                optionally be substituted with one or more substituents                R_(x))—NH—;            -   —COCH₂NH—COCH₂—NH—;            -   —COCH₂NH—;            -   —CONH—(C₁-C₆ alkylene)S—;            -   —CONH—(C₁-C₆ alkylene)NHCO(C₁-C₆ alkylene)S—; and            -   b is 0 or 1, preferably 1.

    -   a drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention, wherein L and (AA)_(w) are as defined in the        preferred definitions for said groups above and X is an        extending group selected from the group consisting of:        -   where D is conjugated via an amine group (for example where            Z is —NH—):            -   —COO—(C₂-C₄ alkylene)NH—;            -   —COO—CH₂-phenylene-NH—, wherein said phenylene group may                optionally be substituted with from one to four                substituents R_(x) selected from the group consisting of                alkyl groups having from 1 to 6 carbon atoms, alkoxy                groups having from 1 to 6 carbon atoms, halogen atoms,                nitro groups and cyano groups;            -   —COO—(C₂-C₄ alkylene)NH—COO—CH₂-(phenylene which may                optionally be substituted with from one to four                substituents R_(x) selected from the group consisting of                alkyl groups having from 1 to 6 carbon atoms, alkoxy                groups having from 1 to 6 carbon atoms, halogen atoms,                nitro groups and cyano groups)-NH—;            -   —COCH₂NH—COCH₂—NH—;            -   —COO—(C₂-C₄ alkylene)S—;            -   —COO—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—; or        -   where D is conjugated via an hydroxy group (for example            where Z is —O—):            -   —CONH—(C₂-C₄ alkylene)NH—;            -   —COO—CH₂-phenylene-NH—, wherein said phenylene group may                optionally be substituted with from one to four                substituents R_(x) selected from the group consisting of                alkyl groups having from 1 to 6 carbon atoms, alkoxy                groups having from 1 to 6 carbon atoms, halogen atoms,                nitro groups and cyano groups;            -   —CONH—(C₂-C₄ alkylene)NH—COO—CH₂-(phenylene which may                optionally be substituted with from one to four                substituents R_(x) selected from the group consisting of                alkyl groups having from 1 to 6 carbon atoms, alkoxy                groups having from 1 to 6 carbon atoms, halogen atoms,                nitro groups and cyano groups)-NH—;            -   —COCH₂NH—COCH₂—NH—;            -   —CONH—(C₂-C₄ alkylene)S—;            -   —CONH—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—; and            -   b is 0 or 1, preferably 1.

    -   a drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention, wherein L and (AA)_(w) are as defined in the        preferred definitions for said groups above and X is an        extending group selected from the group consisting of:        -   where D is conjugated via an amine group (for example where            Z is —NH—):        -   —COO—CH₂-phenylene-NH—        -   —COO(CH₂)₃NHCOOCH₂-phenylene-NH—;        -   —COO(CH₂)₃NH—;        -   —COO(CH₂)₃—S—;        -   —COO(CH₂)₃NHCO(CH₂)₂—; or        -   where D is conjugated via an hydroxy group (for example            where Z is —O—):        -   —COO—CH₂-phenylene-NH—        -   —CONH(CH₂)₃NHCOOCH₂-phenylene-NH—;        -   —CONH(CH₂)₃NH—;        -   —CONH(CH₂)₃—S—;        -   —CONH(CH₂)₃NHCO(CH₂)₂—; and

    -   b is 0 or 1, preferably 1.

    -   a drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention, wherein L, (AA)_(w), and (X)_(b) are as        defined in the preferred definitions for said groups above and T        is an extending group selected from the group consisting of:        -   —CO—(C₁-C₆ alkylene)-NH—;        -   —CO—(C₁-C₆ alkylene)-[O—(C₂-C₆ alkylene)]_(j)-NH—;        -   —COO—(C₁-C₆ alkylene)-[O—(C₂-C₆ alkylene)]_(j)-NH—;        -   where j is an integer from 1 to 25, and

    -   g is 0 or 1.

    -   A drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention, wherein L, (AA)_(w), and (X)_(b) are as        defined in the preferred definitions for said groups above and T        is an extending group selected from the group consisting of:        -   —CO—(C₁-C₄ alkylene)NH—        -   —CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]j-NH—;        -   —COO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]j-NH—;        -   where j is an integer from 1 to 10; and

    -   g is 0 or 1.

    -   A drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention, wherein L, (AA)_(w), and (X)_(b) are as        defined in the preferred definitions for said groups above and T        is an extending group selected from the group consisting of:        -   —CO—(C₁-C₄ alkylene)NH—        -   —CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—;        -   —COO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—;        -   where j is an integer from 1 to 5; and

    -   g is 0 or 1.

    -   A preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), and        (T)_(g) are as defined above and wherein D is a compound of        formula I, IA, IB, IC, ID, IE, IF, IG, Ia, IAa, IBa, ICa, IDa,        IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb, IGb, (IH),        (IHa) or (IHb), or a pharmaceutically acceptable salt, ester,        solvate, tautomer or stereoisomer thereof, wherein R₁ is CN or        OH in compounds of formula I, IA, IB, IC, ID, IE, IG, Ia, IAa,        IBa, ICa, IDa, IEa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IGb, (IH),        (IHa) or (IHb); R₁ is OH in compounds of formula IF, IFa and        IFb, and more preferably R₁ is CN.

    -   Another preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), and        (T)_(g) are as defined above and wherein D is a compound of        formula I, IA, IB, IC, ID, IE, IF, Ia, IAa, IBa, ICa, IDa, IEa,        IFa, Ib, IAb, IBb, ICb, IDb, IEb, IFb, (IH), (IHa) or (IHb), or        a pharmaceutically acceptable salt, ester, solvate, tautomer or        stereoisomer thereof, wherein R₂ is C(═O)R_(a), wherein R_(a) is        selected from hydrogen and substituted or unsubstituted C₁-C₆        alkyl, wherein the optional substituents are one or more        substituents R_(x), and more preferably R₂ is acetyl.

    -   Another preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), and        (T)_(g) are as defined above and wherein D is a compound of        formula I, IA, IB, IC, ID, IE, IF, IG, Ia, IAa, IBa, ICa, IDa,        IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb, IGb, (IH),        (IHa) or (IHb), or a pharmaceutically acceptable salt, ester,        solvate, tautomer or stereoisomer thereof, wherein R₃ is        hydrogen or a —OR_(b) group in compounds of formula I, IC, ID,        IE, IF, IG, la, ICa, IDa, IEa, IFa, IGa, Ib, ICb, IDb, IEb, IFb,        IGb, (IH), (IHa) or (IHb); R₃ is hydrogen in compounds of        formula IA, IAa, or IAb; R₃ is a —OR_(b) group in compounds of        formula IB, IBa, or IBb, wherein R_(b) is a substituted or        unsubstituted C₁-C₆ alkyl group, wherein the optional        substituents are one or more substituents R_(x), and more        preferably R₃ is hydrogen or methoxy. Most preferably R₃ is        hydrogen.

    -   Another preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), and        (T)_(g) are as defined above and wherein D is a compound of        formula (IH), (IHa) or (IHb), or a pharmaceutically acceptable        salt, ester, solvate, tautomer or stereoisomer thereof, wherein        Y is —NH— or —O—.

    -   Another preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), and        (T)_(g) are as defined above and wherein D is a compound of        formula (IH), (IHa) or (IHb), or a pharmaceutically acceptable        salt, ester, solvate, tautomer or stereoisomer thereof, wherein        Z is —NH— or —O—, and more preferably Z is —NH—.

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), and        (T)_(g) are as defined above and wherein D is a compound of        formula (IHa) or (IHb), or a pharmaceutically acceptable salt,        ester, solvate, tautomer or stereoisomer thereof, wherein:        -   R₁ is —CN or —OH;        -   R₂ is —C(═O)R_(a), wherein R_(a) is selected from hydrogen            and substituted or unsubstituted C₁-C₆ alkyl, wherein the            optional substituents are one or more substituents R_(x);        -   R₃ is hydrogen or a —OR_(b) group wherein R_(b) is a            substituted or unsubstituted C₁-C₆ alkyl group, wherein the            optional substituents are one or more substituents R_(x),        -   Y is —NH— or —O—; and        -   Z is —NH— or —O—.

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), and        (T)_(g) are as defined above and wherein D is a compound of        formula (IHa) or (IHb), or a pharmaceutically acceptable salt,        ester, solvate, tautomer or stereoisomer thereof, wherein:        -   R₁ is —CN or —OH;        -   R₂ is acetyl;        -   R₃ is hydrogen or methoxy, more preferably hydrogen;        -   Y is —NH— or —O—; and        -   Z is —NH— or —O—.

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), and        (T)_(g) are as defined above and wherein D is a compound of        formula (IHa) or (IHb), or a pharmaceutically acceptable salt,        ester, solvate, tautomer or stereoisomer thereof, wherein:        -   R₁ is —CN;        -   R₂ is acetyl:        -   R₃ is hydrogen;        -   Y is —NH— or —O—; and        -   Z is —NH—.

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), and        (T)_(g) are as defined above and wherein D is selected from:

-   -   or a pharmaceutically acceptable salt, ester, solvate, tautomer        or stereoisomer thereof; wherein the wavy lines indicate the        point of covalent attachment to (X)_(b) if any, or (AA)_(w) if        any, or to (T)_(g) if any or to (L).    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), and        (T)_(g) are as defined above and wherein D is selected from:

-   -   -   or a pharmaceutically acceptable salt, ester, solvate,            tautomer or stereoisomer thereof; wherein the wavy lines            indicate the point of covalent attachment to (X)_(b) if any,            or (AA)_(w) if any, or to (T)_(g) if any or to (L).

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), (T)_(g)        and D are as defined above and wherein the moiety Ab comprising        at least one antigen binding site is an antigen-binding peptide.

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), (T)_(g)        and D are as defined above and the moiety Ab comprising at least        one antigen binding site is an antibody, a single domain        antibody or an antigen-binding fragment thereof.

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), (T)_(g)        and D are as defined above and the moiety Ab comprising at least        one antigen binding site is a monoclonal, polyclonal antibody or        bispecific antibody and wherein the antibody or antigen-binding        fragment thereof is derived from any species, preferably a        human, mouse or rabbit.

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), (T)_(g)        and D are as defined above and the moiety Ab comprising at least        one antigen binding site is an antibody or antigen-binding        fragment thereof which is selected from the group consisting of        a human antibody, an antigen-binding fragment of a human        antibody, a humanized antibody, an antigen-binding fragment of a        humanized antibody, a chimeric antibody, an antigen-binding        fragment of a chimeric antibody, a glycosylated antibody and a        glycosylated antigen binding fragment.

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), (T)_(g)        and D are as defined above and the moiety Ab comprising at least        one antigen binding site is an antibody or antigen-binding        fragment thereof, wherein the antibody or antigen-binding        fragment thereof is an antigen-binding fragment selected from        the group consisting of an Fab fragment, an Fab′ fragment, an        F(ab′)₂ fragment and an Fv fragment.

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention is one wherein L, (AA)_(w), (X)_(b), (T)_(g)        and D are as defined above and the moiety Ab comprising at least        one antigen binding site is an antibody or antigen-binding        fragment thereof, wherein the antibody or antigen-binding        fragment thereof is a monoclonal antibody which        immunospecifically binds to cancer cell antigens, viral        antigens, antigens of cells that produce autoimmune antibodies        associated with autoimmune disease, microbial antigens, and        preferably a monoclonal antibody which immunospecifically binds        to cancer cell antigens.

    -   A further preferred drug conjugate of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the the        present invention is one wherein L, (AA)_(w), (X)_(b), (T)_(g)        and D are as defined herein and the moiety Ab comprising at        least one antigen binding site is an antibody selected from the        group consisting of Abciximab, Alemtuzumab, Anetumab,        Atezolizumab, Avelumab, Basiliximab, Bevacizumab, Blinatomumab,        Brentuximab, Catumaxomab, Cetuximab, Coltuximab, Daclizumab,        Daratumumab, Denintuzumab, Denosumab, Depatuxizumab,        Dinutuximab, Durvalumab, Elotuzumab, Enfortumab, Glembatumumab,        Gemtuzumab, Ibritumomab, Indatuximab, Indusatumab, Inotuzumab,        Ipilimumab, Labetuzumab, Ladiratuzumab, Laprituximab,        Lifastuzumab, Lorvotuzumab, Milatuzumab, Mirvetuximab,        Naratuximab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab,        Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab,        Pembrolizumab, Pertuzumab, Pinatuzumab, Polatuzumab,        Ramucirumab, Rovalpituzumab, Sacituzumab, Siltuximab,        Sirtratumab, Sofituzumab, Vadastuximab, Vorsetuzumab,        Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an        anti-CD13 antibody and an anti-CD 30 antibody, or an        antigen-binding fragment or an immunologicallly active portion        thereof, wherein preferably the antibody is selected from        Abciximab, Alemtuzumab, Anetumab, Atezolizumab, Avelumab,        Basiliximab, Bevacizumab, Blinatomumab, Brentuximab,        Catumaxomab, Cetuximab, Daclizumab, Daratumumab, Denintuzumab,        Denosumab, Depatuxizumab, Dinutuximab, Durvalumab, Elotuzumab,        Enfortumab, Glembatumumab, Gemtuzumab, Ibritumomab, Indatuximab,        Indusatumab, Inotuzumab, Ipilimumab, Labetuzumab, Ladiratuzumab,        Laprituximab, Mirvetuximab, Naratuximab, Necitumumab,        Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab,        Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab,        Polatuzumab, Ramucirumab, Rovalpituzumab, Sacituzumab,        Siltuximab, Sirtratumab, Vadastuximab, Vorsetuzumab,        Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an        anti-CD13 antibody and an anti-CD antibody, or an        antigen-binding fragment or an immunologicallly active portion        thereof, and yet more preferably Abciximab, Alemtuzumab,        Atezolizumab, Avelumab, Basiliximab, Bevacizumab, Blinatomumab,        Brentuximab, Catumaxomab, Cetuximab, Daclizumab, Daratumumab,        Denosumab, Dinutuximab, Durvalumab, Elotuzumab, Gemtuzumab,        Ibritumomab, Inotuzumab, Ipilimumab, Labetuzumab, Necitumumab,        Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab,        Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab,        Ramucirumab, Rovalpituzumab, Siltuximab, Trastuzumab, an        anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody        and an anti-CD 30 antibody, or an antigen-binding fragment or an        immunologically active portion thereof. Of these, particularly        preferred are Brentuximab, Gemtuzumab, Inozutumab,        Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5        antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or        an antigen-binding fragment or an immunologicallly active        portion thereof; or the antibody is selected from Trastuzumab        and anti-CD13 antibody or an antigen-binding fragment or an        immunologically active portion thereof, particularly Trastuzumab        or an antigen-binding fragment or an immunologicallly active        portion thereof.

    -   Particularly preferred drug conjugates of formula        [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab according to the        present invention include the following:        (a) a drug conjugate according to the present invention wherein:        L is selected from the group consisting of:

wherein:the wavy lines indicate the point of covalent attachments to an Ab (thewavy line to the right) and to (T)_(g) if any, or (AA)_(w) if any, or to(X)_(b) if any, or to (D) (the wavy line to the left);R₁₉ is selected from —C₁-C₁₂ alkylene-, —O—(C₁-C₁₂ alkylene), —C₆-C₁₂arylene in one or more rings which may optionally be substituted withone or more substituents R_(x), —C₁-C₁₂ alkylene-C₆-C₁₂ arylene- whereinthe arylene group is in one or more rings which may optionally besubstituted with one or more substituents R_(x), —C₆-C₁₂ arylene-C₁-C₁₂alkylene- wherein the arylene group is in one or more rings which mayoptionally be substituted with one or more substituents R_(x), —C₅-C₁₂heterocyclo- wherein said heterocyclo group may be a saturated orunsaturated group having one or more rings and comprising at least oneoxygen, nitrogen or sulphur atom in said ring(s), said group optionallybeing substituted with one or more substituents R_(x), —C₁-C₁₂alkylene-(C₅-C₁₂ heterocyclo)- wherein said heterocyclo group may be asaturated or unsaturated group having one or more rings and comprisingat least one oxygen, nitrogen or sulphur atom in said ring(s), saidgroup optionally being substituted with one or more substituents R_(x),—(C₅-C₁₂ heterocyclo)-C₁-C₁₂ alkylene- wherein said heterocyclo groupmay be a saturated or unsaturated group having one or more rings andcomprising at least one oxygen, nitrogen or sulphur atom in saidring(s), said group optionally being substituted with one or moresubstituents R_(x), —(OCH₂CH₂)_(r)— and —CH₂—(OCH₂CH₂)_(r)—, whereineach of the above alkylene substituents whether alone or attached toanother moiety the carbon chain may optionally be substituted by one ormore substituents R_(x);R₃₀ is a —C₁-C₆ alkylene- group;M is selected from the group consisting of —C₁-C₆ alkylene-, —C₁-C₆alkylene-(C₃-C₈ carbocyclo)- and phenylene which may optionally besubstituted with one or more substituents R_(x);r is an integer ranging from 1-6;(AA)_(w) is of formula (II):

wherein the wavy lines indicate the point of covalent attachments to(X)_(b) if any, or to the drug moiety (the wavy line to the left) and to(T)_(g) if any, or to the linker (the wavy line to the right);R₂₁ is, at each occurrence, selected from the group consisting ofhydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

w is an integer ranging from 0 to 12;wherein X is an extending group selected fromwhere D is conjugated via an amine group (for example where Z is —NH—):—COO—(C₁-C₆ alkylene)NH—, —COO—CH₂-(phenylene which may optionally besubstituted with one or more substituents R_(x))—NH—, —COO—(C₁-C₆alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withone or more substituents R_(x))—NH—, —COCH₂NH—COCH₂—NH—, —COCH₂—NH—,—COO—(C₁-C₆ alkylene)S—, —COO—(C₁-C₆ alkylene)NHCO(C₁-C₆ alkylene)S—; orwhere D is conjugated via an hydroxy group (for example where Z is —O—):—CONH—(C₁-C₆ alkylene)NH—, —COO—CH₂-(phenylene which may optionally besubstituted with one or more substituents R_(x))—NH—, —CONH—(C₁-C₆alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withone or more substituents R_(x))—NH—, —COCH₂NH—COCH₂—NH—, —COCH₂NH—,—CONH—(C₁-C₆ alkylene)S—, and —CONH—(C₁-C₆ alkylene)NHCO(C₁-C₆alkylene)S—;b is 0 or 1, preferably 1;wherein T is an extending group selected from —CO—(C₁-C₆ alkylene)-NH—,—CO—(C₁-C₆ alkylene)-[O—(C₂-C₆ alkylene)]_(j)-NH—, and —COO—(C₁-C₆alkylene)-[O—(C₂-C₆ alkylene)]_(j)-NH—, where j is an integer from 1 to25;g is 0 or 1;D is a drug moiety of formula I, IA, IB, IC, ID, IE, IF, IG, IH, la,IAa, IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb,IGb, (IHa) or (IHb), or a pharmaceutically acceptable salt, ester,solvate, tautomer or stereoisomer thereof wherein:R₂ is C(═O)R_(a), in compounds of formuma I, IA, IB, IC, ID, IE, IF, Ia,IAa, IBa, ICa, IDa, IEa, IFa, Ib, IAb, IBb, ICb, IDb, IEb, IFb, (IH),(IHa) or (IHb); R₂ is acetyl in compounds of formula IG, IGa or IGb,wherein R_(a) is selected from hydrogen and substituted or unsubstitutedC₁-C₆ alkyl, wherein the optional substituents are one or moresubstituents R_(x);R₃ is hydrogen or a —OR_(b) group in compounds of formula I, IC, ID, IE,IF, IG, Ia, ICa, IDa, IEa, IFa, IGa, Ib, ICb, IDb, IEb, IFb, IGb, (IH),(IHa) or (IHb); R₃ is hydrogen in compounds of formula IA, IAa, or IAb;R₃ is a —OR_(b) group in compounds of formula IB, IBa, or IBb, whereinR_(b) is a substituted or unsubstituted C₁-C₆ alkyl group, wherein theoptional substituents are one or more substituents R_(x);the moiety Ab comprising at least one antigen binding site is anantibody or an antigen-binding fragment thereof and it is selected fromthe group consisting of a human antibody, an antigen-binding fragment ofa human antibody, a humanized antibody, an antigen-binding fragment of ahumanized antibody, a chimeric antibody, an antigen-binding fragment ofa chimeric antibody, a glycosylated antibody and a glycosylated antigenbinding fragment; andn is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] to themoiety Ab comprising at least one antigen binding site and is in therange from 1 to 12.(b) a drug conjugate according to the present invention selected fromthe formulas (IV), (V) and (VI):

wherein:R₁₉ is selected from —C₁-C₈ alkylene-, —O—(C₁-C₈ alkylene), —C₁-C₈alkylene-C₆-C₁₂ arylene-wherein the arylene group is in one or morerings which may optionally be substituted with one or more substituentsR_(x) and —C₆-C₁₂ arylene-C₁-C₈ alkylene- wherein the arylene group isin one or more rings which may optionally be substituted with one ormore substituents R_(x),wherein each of the above alkylene substituents whether alone orattached to another moiety the carbon chain may optionally besubstituted by one or more substituents R_(x);R₃₀ is a —C₂-C₄ alkylene- group;M is selected from the group consisting of —C₁-C₃ alkylene- and —C₁-C₃alkylene-(C₅-C₇ carbocyclo)-;(AA)_(w) is of formula (II)

wherein:the wavy lines indicate the point of covalent attachments to (X)_(b) ifany, or to the drug moiety (the wavy line to the left) and to (T)_(g) ifany, or to the linker (the wavy line to the right);R₂₁ is, at each occurrence, selected from the group consisting ofhydrogen, methyl, isopropyl, sec-butyl, benzyl, indolylmethyl,—(CH₂)₃NHCONH₂, —(CH₂)₄NH₂, —(CH₂)₃NHC(═NH)NH₂ and —(CH₂)₄NHC(═NH)NH₂;w is an integer from 0 to 6;X is an extending group selected from the group consisting ofwhere D is conjugated via an amine group (for example where Z is —NH—):—COO—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH—, wherein said phenylenegroup may optionally be substituted with from one to four substituentsR_(x) selected from the group consisting of alkyl groups having from 1to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,halogen atoms, nitro groups and cyano groups, —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —COO—(C₂-C₄ alkylene)S—, and—COO—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—; orwhere D is conjugated via an hydroxy group (for example where Z is —O—):—CONH—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH—, wherein saidphenylene group may optionally be substituted with from one to foursubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, halogen atoms, nitro groups and cyano groups, —CONH—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —CONH—(C₂-C₄ alkylene)S—, and—CONH—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—;b is 0 or 1, preferably 1;wherein T is an extending group selected from —CO—(C₁-C₄ alkylene)-NH—,—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to10;g is 0 or 1;D is a drug moiety of formula I, IA, IB, IC, ID, IE, IF, IG, IH, la,IAa, IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb,IGb, (IHa) or (IHb), or a pharmaceutically acceptable salt, ester,solvate, tautomer or stereoisomer thereof wherein:R₂ is acetyl;R₃ is hydrogen or methoxy in compounds of formula I, IC, ID, IE, IF, IG,Ia, ICa, IDa, IEa, IFa, IGa, Ib, ICb, IDb, IEb, IFb, IGb, (IH), (IHa) or(IHb); R₃ is hydrogen in compounds of formula IA, IAa, or IAb; R₃ is amethoxy group in compounds of formula IB, IBa, or IBb, preferably R₃ ishydrogen;the moiety Ab comprising at least one antigen binding site is anantibody or an antigen-binding fragment thereof, wherein the antibody orantigen-binding fragment is a monoclonal antibody whichimmunospecifically binds to cancer cell antigens, viral antigens,antigens of cells that produce autoimmune antibodies associated withautoimmune disease, microbial antigens, and preferably a monoclonalantibody which immunospecifically binds to cancer cell antigens; andn is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein Lis as defined in formulas (IV), (V) or (VI) to the moiety Ab comprisingat least one antigen binding site and is in the range from 3 to 8.(c) a drug conjugate according to the present invention selected fromthe formulas (IV), (V) and (VI):

wherein:R₁₉ is selected from —C₁-C₆ alkylene-, -phenylene-C₁-C₆ alkylene-wherein the phenylene group may optionally be substituted with one ormore substituents R_(x) selected from the group consisting of alkylgroups having from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6carbon atoms, halogen atoms, nitro groups and cyano groups, wherein eachof the above alkylene substituents whether alone or attached to anothermoiety in the carbon chain may optionally be substituted by one or moresubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, aryl groups having from 6 to 12 carbon atoms, halogen atoms,nitro groups and cyano groups, and preferably R₁₉ is a C₁-C₆ alkylenegroup;R₃₀ is a —C₂-C₄ alkylene- group;M is —C₁-C₃ alkylene-(C₅-C₇carbocyclo)-;w is 0 or 2, and where w is 2, then (AA)_(w) is of formula (III):

wherein the wavy lines indicate the point of covalent attachments to(X)_(b) if any, or to the drug moiety (the wavy line to the left) and to(T)_(g) if any, or to the linker (the wavy line to the right);R₂₂ is selected from methyl, benzyl, isopropyl, sec-butyl andindolylmethyl;R₂₃ is selected from methyl, —(CH₂)₄NH₂, —(CH₂)₃NHCONH₂ and—(CH₂)₃NHC(═NH)NH₂;X is an extending group selected from the group consisting of—COO—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH—, wherein said phenylenegroup may optionally be substituted with from one to four substituentsR_(x) selected from the group consisting of alkyl groups having from 1to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,halogen atoms, nitro groups and cyano groups, —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups or cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —COO—(C₂-C₄ alkylene)S—, and—COO—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—;b is 0 or 1, preferably 1;wherein T is an extending group selected from —CO—(C₁-C₄ alkylene)-NH—,—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to5;g is 0 or 1;D is a drug moiety of formula I, IA, IC, ID, IE, IG, IH, la, IAa, ICa,IDa, IEa, IGa, Ib, IAb, ICb, IDb, IEb, IGb, (IHa) or (IHb), or apharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof wherein:

R₁ is CN;

R₂ is acetyl:R₃ is hydrogen;

Y is —NH— or —O—; Z is —NH—;

-   -   the moiety Ab comprising at least one antigen binding site is a        monoclonal antibody selected from the group consisting of        Abciximab, Alemtuzumab, Anetumab, Atezolizumab, Avelumab,        Basiliximab, Bevacizumab, Blinatomumab, Brentuximab,        Catumaxomab, Cetuximab, Coltuximab, Daclizumab, Daratumumab,        Denintuzumab, Denosumab, Depatuxizumab, Dinutuximab, Durvalumab,        Elotuzumab, Enfortumab, Glembatumumab, Gemtuzumab, Ibritumomab,        Indatuximab, Indusatumab, Inotuzumab, Ipilimumab, Labetuzumab,        Ladiratuzumab, Laprituximab, Lifastuzumab, Lorvotuzumab,        Milatuzumab, Mirvetuximab, Naratuximab, Necitumumab,        Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab,        Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab,        Pinatuzumab, Polatuzumab, Ramucirumab, Rovalpituzumab,        Sacituzumab, Siltuximab, Sirtratumab, Sofituzumab, Vadastuximab,        Vorsetuzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5        antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or        an antigen-binding fragment or an immunologicallly active        portion thereof, wherein preferably the antibody is selected        from Abciximab, Alemtuzumab, Anetumab, Atezolizumab, Avelumab,        Basiliximab, Bevacizumab, Blinatomumab, Brentuximab,        Catumaxomab, Cetuximab, Daclizumab, Daratumumab, Denintuzumab,        Denosumab, Depatuxizumab, Dinutuximab, Durvalumab, Elotuzumab,        Enfortumab, Glembatumumab, Gemtuzumab, Ibritumomab, Indatuximab,        Indusatumab, Inotuzumab, Ipilimumab, Labetuzumab, Ladiratuzumab,        Laprituximab, Mirvetuximab, Naratuximab, Necitumumab,        Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab,        Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab,        Polatuzumab, Ramucirumab, Rovalpituzumab, Sacituzumab,        Siltuximab, Sirtratumab, Vadastuximab, Vorsetuzumab,        Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an        anti-CD13 antibody and an anti-CD antibody, or an        antigen-binding fragment or an immunologicallly active portion        thereof, and yet more preferably Abciximab, Alemtuzumab,        Atezolizumab, Avelumab, Basiliximab, Bevacizumab, Blinatomumab,        Brentuximab, Catumaxomab, Cetuximab, Daclizumab, Daratumumab,        Denosumab, Dinutuximab, Durvalumab, Elotuzumab, Gemtuzumab,        Ibritumomab, Inotuzumab, Ipilimumab, Labetuzumab, Necitumumab,        Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab,        Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab,        Ramucirumab, Rovalpituzumab, Siltuximab, Trastuzumab, an        anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody        and an anti-CD 30 antibody, or an antigen-binding fragment or an        immunologically active portion thereof. Of these, particularly        preferred are Brentuximab, Gemtuzumab, Inozutumab,        Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5        antibody, an anti-CD13 antibody and an anti-CD 30 antibody, or        an antigen-binding fragment or an immunologicallly active        portion thereof; or the antibody is selected from Trastuzumab        and anti-CD13 antibody or an antigen-binding fragment or an        immunologically active portion thereof, particularly Trastuzumab        or an antigen-binding fragment or an immunologicallly active        portion thereof; and        n is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]        wherein L is as defined in formulas (IV), (V) or (VI) to the        moiety Ab comprising at least one antigen binding site and is in        the range from 3 to 5.        (d) A drug conjugate according to the present invention selected        from the formulas (IV), (V) and (VI):

wherein:R₁₉ is —C₂-C₆ alkylene-;R₃₀ is a —C₂-C₄ alkylene-;M is —C₁-C₃ alkylene-(C₅-C₇carbocyclo)-;w is 0 or 2, and where w is 2, then (AA)_(w) is of formula (III):

wherein R₂₂ is isopropyl, R₂₃ is selected from methyl and—(CH₂)₃NHCONH₂, wherein the wavy lines indicate the point of covalentattachments to (X)_(b) if any, or to the drug moiety (the wavy line tothe left) and to (T)_(g) if any, or to the linker (the wavy line to theright);X is an extending group selected from the group consisting of—COO—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH—, wherein said phenylenegroup may optionally be substituted with from one to four substituentsR_(x) selected from the group consisting of alkyl groups having from 1to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,halogen atoms, nitro groups and cyano groups, —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —COO—(C₂-C₄ alkylene)S—, and—COO—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—;b is 0 or 1, preferably 1; wherein T is an extending group selected from—CO—(C₁-C₄ alkylene)-NH—, —CO—(C₁-C₄ alkylene)-[0-(C₂-C₄alkylene)]_(j)-NH—, and —COO—(C₁-C₄ alkylene)-[O—(C₂-C₄alkylene)]_(j)-NH—, where j is an integer from 1 to 5;g is 0 or 1;D is a drug moiety selected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof; wherein the wavy line indicates the point ofcovalent attachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g)if any, or to (L);the moiety Ab comprising at least one antigen binding site is selectedfrom Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab,an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and ananti-CD 30 antibody, or an antigen-binding fragment or animmunologicallly active portion thereof, and more preferably its isselected from Trastuzumab and anti-CD13 antibody or an antigen-bindingfragment or an immunologically active portion thereof, particularlyTrastuzumab or an antigen-binding fragment or an immunologicallly activeportion thereof; andn is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein Lis as defined in formulas (IV), (V) or (VI) to the moiety Ab comprisingat least one antigen binding site and is in the range from 3 to 5.(e) A drug conjugate according to the present invention selected fromthe formulas (IV), (V), and (VI):

wherein:R₁₉ is —C₂-C₆ alkylene-;R₃₀ is —C₂-C₄ alkylene-;M is —C₁-C₃ alkylene-(C₅-C₇carbocyclo)-;w is 0 or 2, and where w is 2, then (AA)_(w) is of formula (III):

wherein R₂₂ is isopropyl, R₂₃ is selected from methyl and—(CH₂)₃NHCONH₂, and the wavy lines indicate the point of covalentattachments to (X)_(b) if any, or to the drug moiety (the wavy line tothe left) and to (T)_(g) if any, or to the linker (the wavy line to theright);X is an extending group selected from the group consisting of—COO—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH—, wherein said phenylenegroup may optionally be substituted with from one to four substituentsR_(x) selected from the group consisting of alkyl groups having from 1to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,halogen atoms, nitro groups and cyano groups, —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —COO—(C₂-C₄ alkylene)S—, and—COO—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—;b is 0 or 1, preferably 1;wherein T is an extending group selected from —CO—(C₁-C₄ alkylene)-NH—,—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to5;g is 0 or 1;D is a drug moiety selected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof; wherein the wavy line indicates the point ofcovalent attachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g)if any, or to (L);the moiety Ab comprising at least one antigen binding site is selectedfrom Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab,an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and ananti-CD 30 antibody, or an antigen-binding fragment or animmunologicallly active portion thereof, and more preferably its isselected from Trastuzumab and anti-CD13 antibody or an antigen-bindingfragment or an immunologically active portion thereof, particularlyTrastuzumab or an antigen-binding fragment or an immunologicallly activeportion thereof; andn is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein Lis as defined in formulas (IV), (V) or (VI) to the moiety comprising atleast one antigen binding site and is in the range from 3 to 5.(f) A drug conjugate according to the present invention of formula (IV):

wherein:R₁₉ is C₂-C₅ alkylene-;w is 0 or 2, and where w is 2, then (AA)_(w) is of formula (III):

wherein R₂₂ is isopropyl, R₂₃ is selected from methyl and—(CH₂)₃NHCONH₂, and the wavy lines indicate the point of covalentattachments to (X)_(b) (the wavy line to the left) and to (T)_(g) ifany, or to the linker (the wavy line to the right); andX is a —COOCH₂-phenylene-NH group;b is 1;T is an extending group of formula —CO—(C₁-C₄ alkylene)-[O—(C₂-C₄alkylene)]₄-NH—;g is 0 or 1;or of formula (V)

wherein M is -methyl-cyclohexylene-;b is 1;w is 0;X is an extending group selected from —(CH₂)₃S— and —(CH₂)₃NHCO(CH₂)₂S—g is 0;or of formula (VI)

wherein R₁₉ is —C₂-C₅ alkylene-;R₃₀ is —C₃ alkylene-;w is 0 or 2, and where w is 2, then (AA)_(w) is of formula (III):

wherein R₂₂ is isopropyl, R₂₃ is selected from methyl and—(CH₂)₃NHCONH₂, and the wavy lines indicate the point of covalentattachments to (X)_(b) (the wavy line to the left) and to (T)_(g) ifany, or to the linker (the wavy line to the right); andX is a —COOCH₂-phenylene-NH group;b is 1;T is an extending group of formula —CO—(C₁-C₄ alkylene)-[O—(C₂-C₄alkylene)]₄-NH—;g is 0 or 1;D is a drug moiety selected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof; wherein the wavy line indicates the point ofcovalent attachment to (X)_(b);the moiety Ab comprising at least one antigen binding site isBrentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, ananti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and ananti-CD 30 antibody, or an antigen-binding fragment or animmunologicallly active portion thereof, and more preferably its isselected from Trastuzumab and anti-CD13 antibody or an antigen-bindingfragment or an immunologically active portion thereof, particularlyTrastuzumab or an antigen-binding fragment or an immunologicallly activeportion thereof; andn is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein Lis as defined in formula (IV) to the moiety Ab comprising at least oneantigen binding site and is in the range from 3 to 5, and preferably 4.g) an antibody drug conjugate according according to the presentinvention, selected from the group consisting of:

wherein n is from 2 to 6, more preferably 3, 4, or 5 and each

and

is independently selected from Brentuximab, Gemtuzumab, Inozutumab,Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody,an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-bindingfragment or an immunologically active portion thereof, and morepreferably its is selected from Trastuzumab and anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof,particularly Trastuzumab or an antigen-binding fragment or animmunologically active portion thereof.In an embodiment, antibody drug conjugates according to the presentinvention excludes:

More preferably the antibody drug conjugate is selected from the groupconsisting of:

wherein n is from 2 to 6, more preferably 3, 4, or 5 and

is selected from Trastuzumab and an anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof,more preferably is Trastuzumab or an antigen binding fragment or animmunologically active portion thereof,

wherein n is from 2 to 6, more preferably 3, 4, or 5 and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is from 2 to 6, more preferably 3, 4, or 5 and

is selected from Trastuzumab and an anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof,more preferably is Trastuzumab or an antigen-binding fragment or animmunologically active portion thereof,

wherein n is from 2 to 6, more preferably 3, 4, or 5 and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is from 2 to 6, more preferably 3, 4, or 5 and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof, and

wherein n is from 2 to 6, more preferably 3, 4, or 5 and

is selected from Trastuzumab and an anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof,more preferably is Trastuzumab or an antigen-binding fragment or animmunologically active portion thereof.h) an antibody drug conjugate according to the present invention,selected from the group consisting of:

wherein n is from 2 to 6, more preferably 3, 4, or 5 and each

and

is independently selected from Brentuximab, Gemtuzumab, Inozutumab,Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody,an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-bindingfragment or an immunologically active portion thereof, and morepreferably its is selected from Trastuzumab and anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof,particularly Trastuzumab or an antigen-binding fragment or animmunologically active portion thereof; or

wherein n is from 2 to 6, more preferably 3, 4, or 5 and

is an anti-CD13 antibody or an antigen-binding fragment or animmunologically active portion thereof.More preferably the antibody drug conjugate is selected from the groupconsisting of:

wherein n is from 2 to 6, more preferably 3, 4, or 5 and

is an anti-CD13 antibody or an antigen-binding fragment or animmunologically active portion thereof.

wherein n is from 2 to 6, more preferably 3, 4, or 5 and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is form 2 to 6, more preferably 3, 4, or 5 and and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is form 2 to 6, more preferably 3, 4, or 5 and and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is form 2 to 6, more preferably 3, 4, or 5 and and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is form 2 to 6, more preferably 3, 4, or 5 and and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is form 2 to 6, more preferably 3, 4, or 5 and and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is form 2 to 6, more preferably 3, 4, or 5 and and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is form 2 to 6, more preferably 3, 4, or 5 and and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is form 2 to 6, more preferably 3, 4, or 5 and and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,

wherein n is form 2 to 6, more preferably 3, 4, or 5 and and

is Trastuzumab or an antigen-binding fragment or an immunologicallyactive portion thereof,Particularly preferably, the antibody drug conjugates according to thepresent invention should be in isolated or purified form.Preferred compounds of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ or offormula D-(X)_(b)-(AA)_(w)-(T)_(g)-H according to the present inventioninclude:

-   -   a compound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ or of        formula D-(X)_(b)-(AA)_(w)-(T)_(g)-H wherein each of D, X, AA,        T, Li, b, g and w are as defined herein in the present        invention; but further wherein if the compound is a compound of        formula D-(X)_(b)-(AA)_(w)-(T)_(g)-H then b+w+g≠0.    -   a compound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ or of        formula D-(X)_(b)-(AA)_(w)-(T)_(g)-H according to the present        invention wherein:        L₁ is a linker of formula:

wherein:the wavy line indicates the point of covalent attachment to (T)_(g) ifany, or (AA)_(w) if any, or to (X)_(b) if any, or to D;R₁₉ is selected from —C₁-C₁₂ alkylene-, —O—(C₁-C₁₂ alkylene), —C₆-C₁₂arylene in one or more rings which may optionally be substituted withone or more substituents R_(x), —C₁-C₁₂ alkylene-C₆-C₁₂ arylene- whereinthe arylene group is in one or more rings which may optionally besubstituted with one or more substituents R_(x), —C₆-C₁₂ arylene-C₁-C₁₂alkylene- wherein the arylene group is in one or more rings which mayoptionally be substituted with one or more substituents R_(x), —C₅-C₁₂heterocyclo- wherein said heterocyclo group may be a saturated orunsaturated group having one or more rings and comprising at least oneoxygen, nitrogen or sulphur atom in said ring(s), said group optionallybeing substituted with one or more substituents R_(x), —C₁-C₁₂alkylene-(C₅-C₁₂ heterocyclo)- wherein said heterocyclo group may be asaturated or unsaturated group having one or more rings and comprisingat least one oxygen, nitrogen or sulphur atom in said ring(s), saidgroup optionally being substituted with one or more substituents R_(x),—(C₅-C₁₂ heterocyclo)-C₁-C₁₂ alkylene- wherein said heterocyclo groupmay be a saturated or unsaturated group having one or more rings andcomprising at least one oxygen, nitrogen or sulphur atom in saidring(s), said group optionally being substituted with one or moresubstituents R_(x), —(OCH₂CH₂)_(r) and —CH₂—(OCH₂CH₂)_(r), wherein eachof the above alkylene substituents whether alone or attached to anothermoiety the carbon chain may optionally be substituted by one or moresubstituents R_(x);r is an integer ranging from 1-6; andeach of D, R_(x), X, AA, T, b, g and w is as defined in the presentinvention; but wherein if the compound is a compound of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-H then b+w+g≠0.

-   -   a compound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ or of        formula D-(X)_(b)-(AA)_(w)-(T)_(g)-H according to the present        invention wherein:        L₁ is linker of formula:

wherein:the wavy line indicates the point of covalent attachment to (T)_(g) ifany, or (AA)_(w) if any, or to (X)_(b) if any, or to D;R₁₉ is selected from —C₁-C₈ alkylene-, —O—(C₁-C₈ alkylene), —C₁-C₈alkylene-C₆-C₁₂ arylene-wherein the arylene group is in one or morerings which may optionally be substituted with one or more substituentsR_(x), and —C₆-C₁₂ arylene-C₁-C₈ alkylene- wherein the arylene group isin one or more rings which may optionally be substituted with one ormore substituents R_(x), wherein each of the above alkylene substituentswhether alone or attached to another moiety the carbon chain mayoptionally be substituted by one or more substituents R_(x);(AA)_(w) is of formula (II):

wherein the wavy lines indicate the point of covalent attachments to(X)_(b), if any, or to D (the wavy line to the left) and to (T)_(g) ifany, or L₁ or to a hydrogen atom (the wavy line to the right);wherein R₂₁ is selected, at each occurrence, from the group consistingof hydrogen, methyl, isopropyl, sec-butyl, benzyl, indolylmethyl,—(CH₂)₃NHCONH₂, —(CH₂)₄NH₂, (CH₂)₃NHC(═NH)NH₂ and —(CH₂)₄NHC—(═NH)NH₂,and w is an integer from 0 to 6;X is an extending group selected from the group consisting ofwhere D is conjugated via an amine group (for example where Z is —NH—):—COO—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH, wherein said phenylenegroup may optionally be substituted with from one to four substituentsR_(x) selected from the group consisting of alkyl groups having from 1to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,halogen atoms, nitro groups and cyano groups, —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —COO—(C₂-C₄ alkylene)S—, and—COO—(C₂-C₄ alkylene)-NHCO(C₁-C₃ alkylene)S— orwhere D is conjugated via an hydroxy group (for example where Z is —O—):—CONH—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH—, wherein saidphenylene group may optionally be substituted with from one to foursubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, halogen atoms, nitro groups and cyano groups, —CONH—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —CONH—(C₂-C₄ alkylene)S—, and—CONH—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—;T is an extending group selected from —CO—(C₁-C₄ alkylene)-NH—;—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH— and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to10;b is 0 or 1;g is 0 or 1;wherein if the compound is a compound of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-H then b+w+g≠0; andD is a drug moiety of formula I, IA, IB, IC, ID, IE, IF, IG, Ia, IAa,IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb, and IGb;and is covalently attached via a hydroxy or amine group; oris a drug moiety of formula (IHa) or a formula (IHb), or apharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof wherein:

wherein the wavy lines of (IHa) and (IHb) indicate the point of covalentattachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g) if any,or to Li;

R₁ is —OH or —CN;

R₂ is a —C(═O)R_(a) group, wherein R_(a) is selected from hydrogen andsubstituted or unsubstituted C₁-C₆ alkyl, wherein the optionalsubstituents are one or more substituents R_(x);R₃ is hydrogen or a —OR_(b) group wherein R_(b) is a substituted orunsubstituted C₁-C₆ alkyl group, wherein the optional substituents areone or more substituents R_(x).

Y is —NH— or —O—; and Z is —NH— or —O—.

-   -   a compound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ or of        formula D-(X)_(b)-(AA)_(w)-(T)_(g)-H according to the present        invention wherein:        L₁ is a group of formula:

wherein:the wavy line indicates the point of covalent attachment to (T)_(g) ifany, or (AA)_(w) if any, or to (X)_(b) if any, or to D;R₁₉ is selected from —C₁-C₆ alkylene-, phenylene-C₁-C₆ alkylene- whereinthe phenylene group may optionally be substituted with one or moresubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, halogen atoms, nitro groups and cyano groups, wherein each of theabove alkylene substituents whether alone or attached to another moietyin the carbon chain may optionally be substituted by one or moresubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, aryl groups having from 6 to 12 carbon atoms, halogen atoms,nitro groups and cyano groups, and preferably R₁₉ is a C₁-C₆ alkylenegroup;w is 0 or 2, and where w is 2, then (AA)_(w) is of formula (III):

wherein the wavy lines indicate the point of covalent attachments to X(the wavy line to the left) and to (T)_(g) if any, or L₁ or to ahydrogen atom (the wavy line to the right);R₂₂ is selected from methyl, benzyl, isopropyl, sec-butyl andindolylmethyl;R₂₃ is selected from methyl, —(CH₂)₄NH₂, —(CH₂)₃NHCONH₂ and—(CH₂)₃NHC(═NH)NH₂;X is an extending group selected fromwhere D is conjugated via an amine group (for example where Z is —NH—):—COO—CH₂-phenylene-NH—, —COO(CH₂)₃NHCOO—CH₂-phenylene-NH,—COO—(CH₂)₃)NH—, —COO(CH₂)₃—S—, and —COO—(CH₂)₃NHCO—(CH₂)₂S—, orwhere D is conjugated via an hydroxy group (for example where Z is —O—):—COO—CH₂-phenylene-NH—, —CONH(CH₂)₃NHCOOCH₂-phenylene-NH—,—CONH(CH₂)₃NH—, —CONH(CH₂)₃—S—, and —CONH(CH₂)₃NHCO(CH₂)₂S—.wherein T is an extending group selected from —CO—(C₁-C₄ alkylene)-NH—,—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to5;b is 0 or 1;g is 0 or 1;wherein if the compound is a compound of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-H then b+w+g≠0; andD is a drug moiety of formula I, IA, IB, IC, ID, IE, IF, IG, Ia, IAa,IBa, ICa, IDa, IEa, IFa, IGa, Ib, IAb, IBb, ICb, IDb, IEb, IFb, and IGb;and is covalently attached via a hydroxy or amine group; oris a drug moiety of formula (IHa) or a formula (IHb), or apharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof:

wherein the wavy lines of (IHa) and (IHb) indicate the point of covalentattachment;

R₁ is —CN or —OH;

R₂ is acetyl;R₃ is hydrogen or methoxy, preferably hydrogen;

Y is —NH— or —O—. and Z is —NH— or —O—.

-   -   a compound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ or of        formula D-(X)_(b)-(AA)_(w)-(T)_(g)-H according to the present        invention wherein:        L₁ is a linker of formula:

wherein:the wavy line indicates the point of covalent attachment to (T)_(g) ifany, or (AA)_(w) if any, or to (X)_(b) if any, or to D;R₁₉ is —C₂-C₆ alkylene-;w is 0 or 2, and where w is 2, then (AA)_(w) is of formula (III):

R₂₂ is isopropyl, R₂₃ is selected from methyl and —(CH₂)₃NHCONH₂,wherein the wavy lines indicate the point of covalent attachments to X(the wavy line to the left) and to (T)_(g) if any, or L₁ or to ahydrogen atom (the wavy line to the right);X is an extending group selected from —COO—CH₂-phenylene-NH—,—COO(CH₂)₃NHCOO—CH₂-phenylene-NH, —COO—(CH₂)₃)NH—, —COO(CH₂)₃—S—, and—COO—(CH₂)₃NHCO—(CH₂)₂—;wherein T is an extending group selected from —CO—(C₁-C₄ alkylene)-NH—,—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to5;b is 0 or 1;g is 0 or 1;wherein if the compound is a compound of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-H then b+w+g≠0; andD is a drug moiety selected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof; wherein the wavy line indicates the point ofcovalent attachment.

-   -   a compound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ or of        formula D-(X)_(b)-(AA)_(w)-(T)_(g)-H according to the present        invention wherein:        L₁ is a group of formula:

wherein:the wavy line indicates the point of covalent attachment to (T)_(g) ifany, or (AA)_(w) if any, or to (X)_(b), if any or to D;R₁₉ is a —C₂-C₅ alkylene-;w is 0 or 2, and where w is 2, then (AA)_(w) is of formula (III):

wherein R₂₂ is isopropyl, R₂₃ is selected from methyl and—(CH₂)₃NHCONH₂, wherein the wavy lines indicate the point of covalentattachments to X (the wavy line to the left) and to (T)_(g) if any, orL₁ or to a hydrogen atom (the wavy line to the right);X is a —COO—CH₂-phenylene-NH— group;T is a —CO—(CH₂)₂-[O—(CH₂)₂]₄-NH— group;b is 0 or 1;g is 0 or 1;wherein if the compound is a compound of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-H then b+w+g≠0; and D is a drug moietyselected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof; wherein the wavy line indicates the point ofcovalent attachment.

-   -   a compound of formula D-X-(AA)_(w)-(T)_(g)-L₁ selected from:

-   -   a compound of formula D-X-(AA)_(w)-(T)_(g)-L₁ selected from:

The term “pharmaceutically acceptable salts, esters, solvates, tautomersor stereoisomers” in the drug conjugates of the present invention refersto any pharmaceutically acceptable salt, ester, solvate, hydrate orstereosiomeric form or any other compound which, upon administration tothe patient is capable of providing a compound as described herein,whether directly or indirectly. However, it will be appreciated thatnon-pharmaceutically acceptable salts also fall within the scope of theinvention since those may be useful in the preparation ofpharmaceutically acceptable salts. The preparation of salts, prodrugsand derivatives can be carried out by methods known in the art.

For instance, pharmaceutically acceptable salts of compounds providedherein are synthesized from the parent compound, which contains a basicor acidic moiety, by conventional chemical methods. Generally, suchsalts are, for example, prepared by reacting the free acid or base formsof these compounds with a stoichiometric amount of the appropriate baseor acid in water or in an organic solvent or in a mixture of the two.Generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol or acetonitrile are preferred. Examples of the acid additionsalts include mineral acid addition salts such as, for example,hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate,and organic acid addition salts such as, for example, acetate,trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate,tartrate, malate, mandelate, methanesulphonate and p-toluenesulphonate.Examples of the alkali addition salts include inorganic salts such as,for example, sodium, potassium, calcium and ammonium salts, and organicalkali salts such as, for example, ethylenediamine, ethanolamine,N,N-dialkylenethanolamine, triethanolamine and basic aminoacids salts.

The drug conjugates of the present invention may be in crystalline formeither as free compounds or as solvates (e.g. hydrates) and it isintended that both forms are within the scope of the present invention.Methods of solvation are generally known within the art.

Any compound that is a prodrug of the drug conjugate of the presentinvention is within the scope and spirit of the invention. The term“prodrug” is used in its broadest sense and encompasses thosederivatives that are converted in vivo to the compounds of theinvention. Such derivatives would readily occur to those skilled in theart, and include, for example, compounds where a free hydroxy group isconverted into an ester derivative. Many suitable prodrugs arewell-known to the person in the art and can be found, for example, inBurger “Medicinal Chemistry and Drug Discovery 6^(th) ed. (Donald J.Abraham ed., 2001, Wiley) and “Design and Applications of Prodrugs” (H.Bundgaard ed., 1985, Harwood Academic Publishers), the contents of whichare incorporated herein by reference.

In relations to the compounds of the present invention, thepharmacologically acceptable esters are not particularly restricted, andcan be selected by a person with an ordinary skill in the art. In thecase of said esters, it is preferable that such esters can be cleaved bya biological process such as hydrolysis in vivo. The group constitutingthe said esters (the group shown as R when the esters thereof areexpressed as —COOR) can be, for example, a C₁-C₄ alkoxy C₁-C₄ alkylgroup such as methoxyethyl, 1-ethoxyethyl, 1-methyl-1-methoxyethyl,1-(isopropoxy)ethyl, 2-methoxyethyl, 2-ethoxyethyl,1,1-dimethyl-1-methoxymethyl, ethoxymethyl, propoxymethyl,isopropoxymethyl, butoxymethyl or t-butoxymethyl; a C₁-C₄ alkoxylatedC₁-C₄ alkoxy C₁-C₄ alkyl group such as 2-methoxyethoxymethyl; a C₆-C₁₀aryloxy C₁-C₄ alkyl group such as phenoxymethyl; a halogenated C₁-C₄alkoxy C₁-C₄ alkyl group such as 2,2,2-trichloroethoxymethyl orbis(2-chloroethoxy)methyl; a C₁-C₄ alkoxycarbonyl C₁-C₄ alkyl group suchas methoxycarbonylmethyl; a cyano C₁-C₄ alkyl group such as cyanomethylor 2-cyanoethyl; a C₁-C₄ alkylthiomethyl group such as methylthiomethylor ethylthiomethyl; a C₆-C₁₀ arylthiomethyl group such asphenylthiomethyl or naphthylthiomethyl; a C₁-C₄ alkylsulfonyl C₁-C₄lower alkyl group, which may be optionally substituted with a halogenatom(s) such as 2-methanesulfonylethyl or2-trifluoromethanesulfonylethyl; a C₆-C₁₀ arylsulfonyl C₁-C₄ alkyl groupsuch as 2-benzenesulfonylethyl or 2-toluenesulfonylethyl; a C₁-C₇aliphatic acyloxy C₁-C₄ alkyl group such as formyloxymethyl,acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl,valeryloxymethyl, isovaleryloxymethyl, hexanoyloxymethyl,1-formyloxyethyl, 1-acetoxyethyl, 1-propionyloxyethyl,1-butyryloxyethyl, 1-pivaloyloxyethyl, 1-valeryloxyethyl,1-isovaleryloxyethyl, 1-hexanoyloxyethyl, 2-formyloxyethyl,2-acetoxyethyl, 2-propionyloxyethyl, 2-butyryloxyethyl,2-pivaloyloxyethyl, 2-valeryloxyethyl, 2-isovaleryloxyethyl,2-hexanoyloxyethyl, 1-formyloxypropyl, 1-acetoxypropyl,1-propionyloxypropyl, 1-butyryloxypropyl, 1-pivaloyloxypropyl,1-valeryloxypropyl, 1-isovaleryloxypropyl, 1-hexanoyloxypropyl,1-acetoxybutyl, 1-propionyloxybutyl, 1-butyryloxybutyl,1-pivaloyloxybutyl, 1-acetoxypentyl, 1-propionyloxypentyl,1-butyryloxypentyl, 1-pivaloyloxypentyl or 1-pivaloyloxyhexyl; a C₅-C₆cycloalkylcarbonyloxy C₁-C₄ alkyl group such ascyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl,1-cyclopentylcarbonyloxyethyl, 1-cyclohexylcarbonyloxyethyl,1-cyclopentylcarbonyloxypropyl, 1-cyclohexylcarbonyloxypropyl,1-cyclopentylcarbonyloxybutyl or 1-cyclohexylcarbonyloxybutyl; a C₆-C₁₀arylcarbonyloxy C₁-C₄ alkyl group such as benzoyloxymethyl; a C₁-C₆alkoxycarbonyloxy C₁-C₄ alkyl group such as methoxycarbonyloxymethyl,1-(methoxycarbonyloxy)ethyl, 1-(methoxycarbonyloxy)propyl,1-(methoxycarbonyloxy)butyl, 1-(methoxycarbonyloxy)pentyl,1-(methoxycarbonyloxy)hexyl, ethoxycarbonyloxymethyl,1-(ethoxycarbonyloxy)ethyl, 1-(ethoxycarbonyloxy)propyl,1-(ethoxycarbonyloxy)butyl, 1-(ethoxycarbonyloxy)pentyl,1-(ethoxycarbonyloxy)hexyl, propoxycarbonyloxymethyl,1-(propoxycarbonyloxy)ethyl, 1-(propoxycarbonyloxy)propyl,1-(propoxycarbonyloxy)butyl, isopropoxycarbonyloxymethyl,1-(isopropoxycarbonyloxy)ethyl, 1-(isopropoxycarbonyloxy)butyl,butoxycarbonyloxymethyl, 1-(butoxycarbonyloxy)ethyl,1-(butoxycarbonyloxy)propyl, 1-(butoxycarbonyloxy)butyl,isobutoxycarbonyloxymethyl, 1-(isobutoxycarbonyloxy)ethyl,1-(isobutoxycarbonyloxy)propyl, 1-(isobutoxycarbonyloxy)butyl,t-butoxycarbonyloxymethyl, 1-(t-butoxycarbonyloxy)ethyl,pentyloxycarbonyloxymethyl, 1-(pentyloxycarbonyloxy)ethyl,1-(pentyloxycarbonyloxy)propyl, hexyloxycarbonyloxymethyl,1-(hexyloxycarbonyloxy)ethyl or 1-(hexyloxycarbonyloxy)propyl; a C₅-C₆cycloalkyloxycarbonyloxy C₁-C₄ alkyl group such ascyclopentyloxycarbonyloxymethyl, 1-(cyclopentyloxycarbonyloxy)ethyl,1-(cyclopentyloxycarbonyloxy)propyl, 1-(cyclopentyloxycarbonyloxy)butyl,cyclohexyloxycarbonyloxymethyl, 1-(cyclohexyloxycarbonyloxy)ethyl,1-(cyclohexyloxycarbonyloxy)propyl or 1-(cyclohexyloxycarbonyloxy)butyl;a [5-(C₁-C₄ alkyl)-2-oxo-1,3-dioxolen-4-yl]methyl group such as(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl,(5-ethyl-2-oxo-1,3-dioxolen-4-yl)methyl,(5-propyl-2-oxo-1,3-dioxolen-4-yl)methyl,(5-isopropyl-2-oxo-1,3-dioxolen-4-yl)methyl or(5-butyl-2-oxo-1,3-dioxolen-4-yl)methy; a [5-(phenyl, which may beoptionally substituted with a C₁-C₄ alkyl, C₁-C₄ alkoxy or halogenatom(s))-2-oxo-1,3-dioxolen-4-yl]methyl group such as(5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl,[5-(4-methylphenyl)-2-oxo-1,3-dioxolen-4-yl]methyl,[5-(4-methoxyphenyl)-2-oxo-1,3-dioxolen-4-yl]methyl,[5-(4-fluorophenyl)-2-oxo-1,3-dioxolen-4-yl]methyl or[5-(4-chlorophenyl)-2-oxo-1,3-dioxolen-4-yl]methyl; or a phthalidylgroup, which may be optionally substituted with a C₁-C₄ alkyl or C₁-C₄alkoxy group(s), such as phthalidyl, dimethylphthalidyl ordimethoxyphthalidyl, and is preferably a pivaloyloxymethyl group,phthalidyl group or (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl group, andmore preferably a (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl group.

Any compound referred to herein is intended to represent such specificcompound as well as certain variations or forms. In particular,compounds referred to herein may have asymmetric centres and thereforeexist in different enantiomeric forms. All optical isomers andstereoisomers of the compounds referred to herein, and mixtures thereof,are considered within the scope of the present invention. Thus any givencompound referred to herein is intended to represent any one of aracemate, one or more enantiomeric forms, one or more diastereomericforms, one or more atropisomeric forms, and mixtures thereof.Particularly, the drug conjugates of formula[D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)]_(n)-Ab and compounds of formulaD-X-(AA)_(w)-(T)_(g)-L₁ or D-X-(AA)_(w)-(T)_(g)-H may includeenantiomers depending on their asymmetry or diastereoisomers.Stereoisomerism about the double bond is also possible, therefore insome cases the molecule could exist as (E)-isomer or (Z)-isomer. If themolecule contains several double bonds, each double bond will have itsown stereoisomerism, that could be the same or different than thestereoisomerism of the other double bonds of the molecule. The singleisomers and mixtures of isomers fall within the scope of the presentinvention.

Furthermore, compounds referred to herein may exist as geometric isomers(i.e., cis and trans isomers), as tautomers, or as atropisomers.Specifically, the term tautomer refers to one of two or more structuralisomers of a compound that exist in equilibrium and are readilyconverted from one isomeric form to another. Common tautomeric pairs areamine-imine, amide-imide, keto-enol, lactam-lactim, etc. Additionally,any compound referred to herein is intended to represent hydrates,solvates, and polymorphs, and mixtures thereof when such forms exist inthe medium. In addition, compounds referred to herein may exist inisotopically-labelled forms. All geometric isomers, tautomers,atropisomers, hydrates, solvates, polymorphs, and isotopically labelledforms of the compounds referred to herein, and mixtures thereof, areconsidered within the scope of the present invention.

Protected forms of the compounds disclosed herein are considered withinthe scope of the present invention. Suitable protecting groups are wellknown for the skilled person in the art. A general review of protectinggroups in organic chemistry is provided by Wuts, PGM and Greene TW inProtecting Groups in Organic Synthesis, 4^(th) Ed. Wiley-lnterscience,and by Kocienski P J in Protecting Groups, 3^(rd) Ed. Georg ThiemeVerlag. These references provide sections on protecting groups for OH,amino and SH groups. All these references are incorporated by referencein their entirety.

Within the scope of the present invention an OH protecting group isdefined to be the O-bonded moiety resulting from the protection of theOH through the formation of a suitable protected OH group. Examples ofsuch protected OH groups include ethers, silyl ethers, esters,sulfonates, sulfenates and sulfinates, carbonates, and carbamates. Inthe case of ethers the protecting group for the OH can be selected frommethyl, methoxymethyl, methylthiomethyl,(phenyldimethylsilyl)methoxymethyl, benzyloxymethyl,p-methoxybenzyloxymethyl, [(3,4-dimethoxybenzyl)oxy]methyl,p-nitrobenzyloxymethyl, o-nitrobenzyloxymethyl,[(R)-1-(2-nitrophenyl)ethoxy]methyl, (4-methoxyphenoxy)methyl,guaiacolmethyl, [(p-phenylphenyl)oxy]methyl, t-butoxymethyl,4-pentenyloxymethyl, siloxymethyl, 2-methoxyethoxymethyl,2-cyanoethoxymethyl, bis(2-chloroethoxy)methyl,2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)ethoxymethyl,menthoxymethyl, 0-bis(2-acetoxy-ethoxy)methyl, tetrahydropyranyl,fluorous tetrahydropyranyl, 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl,4-methoxy-tetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)-phenyl]-4-methoxypiperidin-4-yl,1-(2-fluorophenyl)-4-methoxypiperidin-4-yl,1-(4-chlorophenyl)-4-methoxypiperidin-4-yl, 1,4-dioxan-2-yl,tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-hydroxyethyl, 2-bromoethyl,1-[2-(trimethylsilyl)ethoxy]ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,1-methyl-1-phenoxyethyl, 2,2,2-trichloroethyl,1,1-dianisyl-2,2,2-trichloroethyl,1,1,1,3,3,3-hexafluoro-2-phenylisopropyl, 1-(2-cyanoethoxy)ethyl,2-trimethylsilylethyl, 2-(benzylthio)ethyl, 2-(phenylselenyl)ethyl,t-butyl, cyclohexyl, 1-methyl-T-cyclopropylmethyl, allyl, prenyl,cinnamyl, 2-phenallyl, propargyl, p-chlorophenyl, p-methoxyphenyl,p-nitrophenyl, 2,4-dinitrophenyl,2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenyl, benzyl, p-methoxybenzyl,3,4-dimethoxybenzyl, 2,6-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl,pentadienylnitrobenzyl, pentadienylnitropiperonyl, halobenzyl,2,6-dichlorobenzyl, 2,4-dichlorobenzyl, 2,6-difluorobenzyl,p-cyanobenzyl, fluorous benzyl, 4-fluorousalkoxybenzyl,trimethylsilylxylyl, p-phenylbenzyl, 2-phenyl-2-propyl,p-acylaminobenzyl, p-azidobenzyl, 4-azido-3-chlorobenzyl,2-trifluoromethylbenzyl, 4-trifluoromethylbenzyl,p-(methylsulfinyl)benzyl, p-siletanylbenzyl, 4-acetoxybenzyl,4-(2-trimethylsilyl)ethoxymethoxybenzyl, 2-naphthylmethyl, 2-picolyl,4-picolyl, 3-methyl-2-picolyl N-oxide, 2-quinolinylmethyl,6-methoxy-2-(4-methylphenyl)-4-quinolinemethyl, 1-pyrenylmethyl,diphenylmethyl, 4-methoxydiphenylmethyl, 4-phenyldiphenylmethyl,p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl,tris(4-t-butylphenyl)methyl, a-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenyl-methyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxy)phenyldiphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,4,4′-dimethoxy-3″-[N-(imidazolylmethyl)]trityl,4,4′-dimethoxy-3″-[N-(imidazolylethyl)carbamoyl]trityl,bis(4-methoxyphenyl)-1′-pyrenylmethyl,4-(17-tetrabenzo[a,c,g,i]fluorenylmethyl)-4,4″-dimethoxytrityl,9-anthryl, 9-(9-phenyl)xanthenyl, 9-phenylthioxanthyl,9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl,4,5-bis(ethoxycarbonyl)-[1,3]-dioxolan-2-yl, benzisothiazolylS,S-dioxide. In the case of silyl ethers the protecting group for the OHcan be selected from trimethylsilyl, triethylsilyl, triisopropylsilyl,dimethylisopropylsilyl, diethylisopropylsilyl, dimethylhexylsilyl,2-norbornyldimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl,di-t-butylmethylsilyl, bis(f-butyl)-1-pyrenylmethoxysilyl,tris(trimethylsilyl)silyl, (2-hydroxystyryl)dimethylsilyl,(2-hydroxystyryl)diisopropylsilyl, t-butylmethoxyphenylsilyl,t-butoxydiphenylsilyl, 1,1,3,3-tetraisopropyl-3-[2-(triphenylmethoxy)ethoxy]disiloxane-1-yl, and fluorous silyl. In the case of esters theprotecting group for the OH together with the oxygen atom of theunprotected OH to which it is attached form an ester that can beselected from formate, benzoylformate, acetate, chloroacetate,dichloroacetate, trichloroacetate, trichloroacetamidate,trifluoroacetate, methoxyacetate, triphenylmethoxyacetate,phenoxyacetate, p-chlorophenoxyacetate, phenylacetate, diphenylacetate,3-phenylpropionate, bisfluorous chain type propanoyl, 4-pentenoate,4-oxopentanoate, 4,4-(ethylenedithio)pentanoate,5[3-bis(4-methoxyphenyl)hydro-xymethylphenoxy]levulinate, pivaloate,1-adamantoate, crotonate, 4-methoxycrotonate, benzoate,p-phenylbenzoate, 2,4,6-trimethylbenzoate, 4-bromobenzoate,2,5-difluorobenzoate, p-nitrobenzoate, picolinate, nicotinate,2-(azidomethyl)benzoate, 4-azido-butyrate, (2-azidomethyl)phenylacetate,2-{[(tritylthio)oxy]methyl}benzoate,2-{[(4-methoxytritylthio)oxy]methyl}benzoate,2-{[methyl(tritylthio)amino]methyl}benzoate,2-{{[(4-methoxytrityl)thio]methylamino}methyl}benzoate,2-(allyloxy)phenylacetate, 2-(prenyloxymethyl)benzoate,6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoate,6-(levulinyloxymethyl)-3-methoxy-4-nitrobenzoate, 4-benzyloxybutyrate,4-trialkylsilyloxy-butyrate, 4-acetoxy-2,2-dimethylbutyrate,2,2-dimethyl-4-pentenoate, 2-iodobenzoate, 4-nitro-4-methylpentanoate,o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate,4-(methylthio-methoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2-(chloroacetoxymethyl)benzoate, 2-[(2-chloroacetoxy)ethyl]benzoate,2-[2-(benzyloxy)ethyl]benzoate,2-[2-(4-methoxybenzyl-oxy)ethyl]benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenyl-acetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, a-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, and 2-chlorobenzoate. In thecase of sulfonates, sulfenates and sulfinates the protecting group forthe OH together with the oxygen atom of the unprotected OH to which itis attached form a sulfonate, sulfenate or sulfinates that can beselected from sulfate, allylsulfonate, methanesulfonate,benzylsulfonate, tosylate, 2-[(4-nitrophenyl)ethyl]sulfonate,2-trifluoromethylbenzenesulfonate, 4-monomethoxytritylsulfenate, alkyl2,4-dinitrophenylsulfenate,2,2,5,5-tetramethylpyrrolidin-3-one-1-sulfinate, anddimethylphosphinothioyl. In the case of carbonates the protecting groupfor the OH together with the oxygen atom of the unprotected OH to whichit is attached form a carbonate that can be selected from methylcarbonate, methoxymethyl carbonate, 9-fluorenylmethyl carbonate, ethylcarbonate, bromoethyl carbonate, 2-(methylthiomethoxy)ethyl carbonate,2,2,2-trichloroethyl carbonate, 1,1-dimethyl-2,2,2-trichloroethylcarbonate, 2-(trimethylsilyl)ethyl carbonate,2-[dimethyl(2-naphthylmethyl)silyl]ethyl carbonate,2-(phenylsulfonyl)ethyl carbonate, 2-(triphenylphosphonio)ethylcarbonate, c/s-[4-[[(methoxytrityl)sulfenyl]oxy]tetrahydrofuran-3-yl]oxycarbonate, isobutyl carbonate, t-butyl carbonate, vinyl carbonate, allylcarbonate, cinnamyl carbonate, propargyl carbonate, p-chlorophenylcarbonate, p-nitrophenyl carbonate, 4-ethoxy-1-naphthyl carbonate,6-bromo-7-hydroxycoumarin-4-ylmethyl carbonate, benzyl carbonate,o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, anthraquinon-2-ylmethylcarbonate, 2-dansylethyl carbonate, 2-(4-nitrophenyl)ethyl carbonate,2-(2,4-dinitrophenyl)ethyl carbonate, 2-(2-nitrophenyl)propyl carbonate,2-(3,4-methylenedioxy-6-nitrophenyl)propyl carbonate,2-cyano-1-phenylethyl carbonate, 2-(2-pyridyl)amino-1-phenylethylcarbonate, 2-[N-methyl-N-(2-pyridyl)]amino-1-phenylethyl carbonate,phenacyl carbonate, 3′, 5′-dimethoxybenzoin carbonate, methyldithiocarbonate, and S-benzyl thiocarbonate. And in the case ofcarbamates the protecting group for OH together with the oxygen atom ofthe unprotected OH to which it is attached forms a carbamate that can beselected from dimethyl thiocarbamate, N-phenyl carbamate, andN-methyl-N-(o-nitrophenyl) carbamate.

Within the scope of the present invention an amino protecting group isdefined to be the N-bonded moiety resulting from the protection of theamino group through the formation of a suitable protected amino group.Examples of protected amino groups include carbamates, ureas, amides,heterocyclic systems, N-alkyl amines, N-alkenyl amines, N-alkynylamines, N-aryl amines, imines, enamines, N-metal derivatives, N—Nderivatives, N—P derivatives, N—Si derivatives, and N—S derivatives. Inthe case of carbamates the protecting group for the amino group togetherwith the amino group to which it is attached form a carbamate that canbe selected from methyl carbamate, ethyl carbamate, 9-fluorenylmethylcarbamate, 2,6-di-t-butyl-9-fluorenylmethyl carbamate,2,7-bis(trimethylsilyl)fluorenylmethyl carbamate,9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethylcarbamate, 17-tetrabenzo[a,c,g,i]fluorenylmethyl carbamate,2-chloro-3-indenylmethyl carbamate, benz[f]inden-3-ylmethyl carbamate,1,1-dioxobenzo[b]-thiophene-2-ylmethyl carbamate,2-methylsulfonyl-3-phenyl-1-prop-2-enyl carbamate,2,7-di-t-butyl-[9,(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate, 2,2,2-trichloroethyl carbamate, 2-trimethylsilylethylcarbamate, (2-phenyl-2-trimethylsilyl)ethyl carbamate, 2-phenylethylcarbamate, 2-chloroethyl carbamate, 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate,1,1-dimethyl-2,2,2-trichloroethyl carbamate, 2-(2′-pyridyl)ethylcarbamate, 2-(4′-pyridyl)ethyl carbamate, 2,2-bis(4′-nitrophenyl)ethylcarbamate, 2-[(2-nitrophenyl)dithio]-1-phenylethyl carbamate,2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate,fluorous BOC carbamate, 1-adamantyl carbamate, 2-adamantyl carbamate,1-(1-adamantyl)-1-methylethyl carbamate, 1-methyl-1-(4-byphenylyl)ethylcarbamate, 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate,triisopropylsilyloxy carbamate, vinyl carbamate, allyl carbamate, prenylcarbamate, 1-isopropylallyl carbamate, cinnamyl carbamate,4-nitrocinnamyl carbamate, 3-(3′-pyridyl)prop-2-enyl carbamate,hexadienyl carbamate, propargyl carbamate, 1,4-but-2-ynyl biscarbamate,8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate, 3,5-di-t-butylbenzyl carbamate,p-methoxybenzyl carbamate, p-nitrobenzyl carbamate, p-bromobenzylcarbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate,4-methylsulfinylbenzyl carbamate, 4-trifluoromethylbenzyl carbamate,fluorous benzyl carbamate, 2-naphthylmethyl carbamate, 9-anthrylmethylcarbamate, diphenylmethyl carbamate, 4-phenylacetoxybenzyl carbamate,4-azidobenzyl carbamate, 4-azido-methoxybenzyl carbamate,m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)-benzyl carbamate,5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, 2-(4-nitrophenylsulfonyl)ethylcarbamate, 2-(2,4-dinitrophenylsulfonyl)ethyl carbamate,2-(4-trifluoromethylphenylsulfonyl)ethyl carbamate,[2-(1,3-dithianyl)]methyl carbamate, 2-phosphonioethyl carbamate,2-[phenyl(methyl)sulfonio]ethyl carbamate,1-methyl-1-(triphenylphosphonio)ethyl carbamate,1,1-dimethyl-2-cyanoethyl carbamate, 2-dansylethyl carbamate,2-(4-nitrophenyl)ethyl carbamate, 4-methylthiophenyl carbamate,2,4-dimethylthiophenyl carbamate, m-nitrophenyl carbamate,3,5-dimethoxybenzyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethylcarbamate, a-methylnitropiperonyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, 2-nitrophenylethyl carbamate, 6-nitroveratryl carbamate,4-methoxyphenacyl carbamate, 3′, 5′-dimethoxybenzoin carbamate,9-xanthenylmethyl carbamate, N-methyl-N-(o-nitrophenyl) carbamate,t-amyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexylcarbamate, 1-methyl-1-cyclopropylmethyl carbamate, cyclobutyl carbamate,cyclopentyl carbamate, cyclohexyl carbamate, isobutyl carbamate,isobornyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzylcarbamate, diisopropylmethyl carbamate, 2,2-dimethoxy-carbonylvinylcarbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate,1,1-dimethyl-3-(N,N-dimethyl-carboxamido)propyl carbamate, butynylcarbamate, 1,1-dimethylpropynyl carbamate, 2-iodoethyl carbamate,1-methyl-1-(4′-pyridyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, p-(phenylazo)benzyl carbamate,2,4,6-trimethylbenzyl carbamate, isonicotinyl carbamate,4-(trimethyl-ammonium)benzyl carbamate, p-cyanobenzyl carbamate,di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, phenylcarbamate, 2,4,6-tri-t-butylphenyl carbamate, 1-methyl-1-phenylethylcarbamate, and S-benzyl thiocarbamate. In the case of ureas theprotecting groups for the amino group can be selected fromphenothiazinyl-(10)-carbonyl, N′-p-toluenesulfonylaminocarbonyl,N′-phenylaminothiocarbonyl, 4-hydroxyphenylaminocarbonyl,3-hydroxytryptaminocarbonyl, and N′-phenylaminothiocarbonyl. In the caseof amides the protecting group for the amino together with the aminogroup to which it is attached form an amide that can be selected fromformamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide,pent-4-enamide, picolinamide, 3-pyridylcarboxamide,N-benzoylphenylalanyl amide, benzamide, p-phenylbenzamide,o-nitrophenylacetamide, 2,2-dimethyl-2-(o-nitrophenyl)acetamide,o-nitrophenoxyacetamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide, 3-methyl-3-nitrobutanamide,o-nitrocinnamide, o-nitrobenzamide,3-(4-t-butyl-2,6-dinitrophenyl)-2,2-dimethylpropanamide,o-(benzoyloxyme-thyl)benzamide, 2-(acetoxymethyl)benzamide,2-[(f-butyldiphenylsiloxy)methyl]benzamide, 3-(3′, 6′-dioxo-2′, 4′,5′-trimethylcyclohexa-1′, 4′-diene)-3,3-dimethylpropionamide,o-hydroxy-frans-cinnamide, 2-methyl-2-(o-phenylazophenoxy)propanamide,4-chlorobutanamide, aceto-acetamide, 3-(p-hydroxyphenyl)propanamide,(N′-dithiobenzyloxycarbonylamino)acetamide, and N-acetylmethionineamide. In the case of heterocyclic systems the protecting group for theamino group together with the amino group to which it is attached form aheterocyclic system that can be selected from4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dichlorophthalimide,N-tetrachlorophthalimide, N-4-nitrophthalimide, N-thiodiglycoloyl,N-dithiasuccinimide, N-2,3-diphenylmaleimide, N-2,3-dimethylmaleimide,N-2,5-dimethylpyrrole, N-2,5-bis(triisopropylsiloxy)pyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct,N-1,1,3,3-tetramethyl-1,3-disilaisoindoline, N-diphenylsilyldiethylene,N-5-substituted-1,3-dimethyl-1,3,5-triazacyclohexan-2-one,N-5-substituted-1,3-benzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, and 1,3,5-dioxazine. In the case of N-alkyl,N-alkenyl, N-alkynyl or N-aryl amines the protecting group for the aminogroup can be selected from N-methyl, N-t-butyl, N-allyl, N-prenyl,N-cinnamyl, N-phenylallyl, N-propargyl, N-methoxymethyl,N-[2-(trimethylsilyl)ethoxy]methyl, N-3-acetoxypropyl, N-cyanomethyl,N-2-azanorbornenes, N-benzyl, N-4-methoxybenzyl, N-2,4-dimethoxybenzyl,N-2-hydroxybenzyl, N-ferrocenylmethyl, N-2,4-dinitrophenyl,o-methoxyphenyl, p-methoxyphenyl, N-9-phenylfluorenyl, N-fluorenyl,N-2-picolylamine N′-oxide, N-7-methoxycoumar-4-ylmethyl,N-diphenylmethyl, N-bis(4-methoxyphenyl)methyl, N-5-dibenzosuberyl,N-triphenylmethyl, N-(4-methylphenyl)diphenylmethyl, andN-(4-methoxyphenyl)diphenylmethyl. In the case of imines the protectinggroup for the amino group can be selected fromN-1,1-dimethylthiomethylene, N-benzylidene, N-p-methoxybenzylidene,N-diphenylmethylene, N-[2-pyridyl)mesityl]methylene,N—(N′,N′-dimethylaminomethylene), N—(N,N-dibenzylaminomethylene),N—(N′-t-butylaminome-thylene), N,N-isopropylidene, N-p-nitrobenzylidene,N-salicylidene, N-5-chlorosalicylidene,N-(5-chloro-2-hydroxyphenyl)phenylmethylene, N-cyclohexylidene, andN-t-butylidene. In the case of enamines the protecting group for theamino group can be selected from N-(5,5-dimethyl-3-oxo-1-cyclohexenyl),N-2,7-dichloro-9-fluorenylmethylene,N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl,N-(1,3-dimethyl-2,4,6-(1H,3H,5H)-trioxopyrimidine-5-ylidene)-methyl,N-4,4,4-trifluoro-3-oxo-1-butenyl, andN-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl). In the case of N-metalderivatives the protecting group for the amino group can be selectedfrom N-borane, N-diphenylborinic ester, N-diethylborinic ester,N-9-borabicyclononane, N-difluoroborinic ester, and3,5-bis(trifluoromethyl)phenylboronic acid; and also includingN-phenyl(pentacarbonylchromium)carbenyl,N-phenyl(pentacarbonyl-tungsten)carbenyl,N-methyl(pentacarbonylchromium)carbenyl,N-methyl(pentacarbonyltungsten)carbenyl, N-copper chelate, N-zincchelate, and a 18-crown-6-derivative. In the case of N—N derivatives theprotecting group for the amino group together with the amino group towhich it is attached form a N—N derivative that can be selected fromN-nitroamino, N-nitrosoamino, amine N-oxide, azide, triazene derivative,and N-trimethylsilylmethyl-N-benzylhydrazine. In the case of N—Pderivatives the protected group for the amino group together with theamino group to which it is attached form a N—P derivative that can beselected from diphenylphosphinamide, dimethylthiophosphinamide,diphenylthiophosphinamide, dialkyl phosphoramidate, dibenzylphosphoramidate, diphenyl phosphoramidate, andiminotriphenylphosphorane. In the case of N-Si derivatives theprotecting group for the NH₂ can be selected from t-butyldiphenylsilyland triphenylsilyl. In the case of N—S derivatives the protected aminogroup can be selected from N-sulfenyl or N-sulfonyl derivatives. TheN-sulfenyl derivatives can be selected from benzenesulfenamide,2-nitrobenzenesulfenamide, 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfe-namide,1-(2,2,2-trifluoro-1,1-diphenyl)ethylsulfenamide, andN-3-nitro-2-pyridinesulfenamide. The N-sulfonyl derivatives can beselected from methanesulfonamide, trifluoromethanesulfonamide,t-butylsulfonamide, benzylsulfonamide, 2-(trimethylsilyl)ethanesulfonamide, p-toluenesulfonamide, benzenesulfonamide,o-anisylsulfonamide, 2-nitrobenzenesulfonamide,4-nitrobenzenesulfonamide, 2,4-dinitrobenzenesulfonamide,2-naphthalenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide,2-(4-methylphenyl)-6-methoxy-4-methylsulfonamide,9-anthracenesulfonamide, pyridine-2-sulfonamide,benzothiazole-2-sulfonamide, phenacylsulfonamide,2,3,6-trimethyl-4-methoxybenzenesulfonamide,2,4,6-trimethoxybenzenesulfonamide,2,6-dimethyl-4-methoxy-benzenesulfonamide,pentamethylbenzenesulfonamide,2,3,5,6-tetramethyl-4-methoxyben-zenesulfonamide,4-methoxybenzenesulfonamide, 2,4,6-trimethylbenzenesulfonamide,2,6-dimethoxy-4-methylbenzenesulfonamide,3-methoxy-4-t-butylbenzenesulfonamide, and2,2,5,7,8-pentamethylchroman-6-sulfonamide.

Within the scope of the present invention a protecting group for SH isdefined to be the S-bonded moiety resulting from the protection of theSH group through the formation of a suitable a protected SH group.Examples of such protected SH groups include thioethers, disulfides,silyl thioethers, thioesters, thiocarbonates, and thiocarbamates. In thecase of thioethers the protecting group for the SH can be selected fromS-alkyl, S-benzyl, S-p-methoxybenzyl, S-o-hydroxybenzyl,S-p-hydroxybenzyl, S-o-acetoxybenzyl, S-p-acetoxybenzyl,S-p-nitrobenzyl, S-o-nitrobenzyl, S-2,4,6-trimethylbenzyl,S-2,4,6-trimethoxybenzyl, S-4-picolyl, S-2-picolyl-N-oxide,S-2-quinolinylmethyl, S-9-anthrylmethyl, S-9-fluorenylmethyl,S-xanthenyl, S-ferrocenylmethyl, S-diphenylmethyl,S-bis(4-methoxyphenyl)methyl, S-5-dibenzosuberyl, S-triphenylmethyl,4-methoxytrityl, S-diphenyl-4-pyridylmethyl, S-phenyl,S-2,4-dinitrophenyl, S-2-quinolyl, S-t-butyl, S-1-adamantyl,S-methoxymethyl, S-isobutoxymethyl, S-benzyloxymethyl, S-1-ethoxyethyl,S-2-tetrahydropyranyl, S-benzylthiomethyl, S-phenylthiomethyl,S-acetamidomethyl (Acm), S-trimethylacetamidomethyl, S-benzamidomethyl,S-allyloxycarbonylaminomethyl,S—N-[2,3,5,6-tetrafluoro-4-(N′-piperidino)-phenyl-N-allyloxycarbonylaminomethyl,S-phthalimidomethyl, S-phenylacetamidomethyl, S-acetylmethyl,S-carboxymethyl, S-cyanomethyl, S-(2-nitro-1-phenyl)ethyl,S-2-(2,4-dinitrophenyl)ethyl, S-2-(4′-pyridyl)ethyl, S-2-cyanoethyl,S-2-(trimethylsilyl)ethyl, S-2,2-bis(carboethoxy)ethyl,S-(1-m-nitrophenyl-2-benzoyl)ethyl, S-2-phenylsulfonylethyl,S-1-(4-methylphenylsulfonyl)-2-methylprop-2-yl, and S-p-hydroxyphenacyl.In the case of disulfides the protected SH group can be selected fromS-ethyl disulfide, S-t-butyl disulfide, S-2-nitrophenyl disulfide,S-2,4-dinitrophenyl disulfide, S-2-phenylazophenyl disulfide,S-2-carboxyphenyl disulfide, and S-3-nitro-2-pyridyl disulfide. In thecase of silyl thioethers the protecting group for the SH can be selectedfrom the list of groups that was listed above for the protection of OHwith silyl ethers. In the case of thioesters the protecting group forthe SH can be selected from S-acetyl, S-benzoyl, S-2-methoxyisobutyryl,S-trifluoroacetyl,S—N-[[p-biphenylyl)-isopropyloxy]carbonyl]-N-methyl-y-aminothiobutyrate,and S—N-(t-butoxycarbonyl)-N-methyl-y-aminothiobutyrate. In the case ofthiocarbonate protecting group for the SH can be selected fromS-2,2,2-trichloroethoxycarbonyl, S-t-butoxycarbonyl,S-benzyloxycarbonyl, S-p-methoxybenzyloxycarbonyl, andS-fluorenylmethylcarbonyl. In the case of thiocarbamate the protected SHgroup can be selected from S—(N-ethylcarbamate) andS—(N-methoxymethylcarbamate).

The mention of these groups should not be interpreted as a limitation ofthe scope of the invention, since they have been mentioned as a mereillustration of protecting groups for OH, amino and SH groups, butfurther groups having said function may be known by the skilled personin the art, and they are to be understood to be also encompassed by thepresent invention.

To provide a more concise description, some of the quantitativeexpressions given herein are not qualified with the term “about”. It isunderstood that, whether the term “about” is used explicitly or not,every quantity given herein is meant to refer to the actual given value,and it is also meant to refer to the approximation to such given valuethat would reasonably be inferred based on the ordinary skill in theart, including equivalents and approximations due to the experimentaland/or measurement conditions for such given value.

“Antibody-drug-conjugates (ADCs)” represent a targeted strategy todeliver a cytotoxic molecule to a cancer cell (see, for example,International Patent Applications WO-A-2004/010957, WO-A-2006/060533 andWO-A-2007/024536). Such compounds are typically referred to as drug,toxin and radionuclide “conjugates”. Tumor cell killing occurs uponbinding of the drug conjugate to a tumor cell and release and/oractivation of the cytotoxic activity of the drug moiety. The selectivityafforded by drug conjugates minimizes toxicity to normal cells, therebyenhancing tolerability of the drug in the patient. Three examples ofdrug antibody conjugates of this type that have received marketingapproval are: Gemtuzumab ozogamicin for acute myelogenous leukemia,Brentuximab vedotin for relapsed and refractory Hodgkin lymphoma andanaplastic large cell lymphoma, and ado-Trastuzumab emtansine for breastcancer, especially HER2+.

The effectiveness of drugs for cancer chemotherapy generally relies ondifferences in growth rates, biochemical pathways, and physiologicalcharacteristics between cancer and normal tissues. Consequently, moststandard chemotherapeutics are relatively nonspecific and exhibitdose-limiting toxicities that contribute to suboptimal therapeuticeffects. One approach to selectively target malignant cells and nothealthy tissues is to use specific monoclonal antibodies (mAbs) thatrecognize tumor-associated antigens expressed on the surface of tumorcells [Meyer, D. L. & Senter, P. D. (2003) Recent advances in antibodydrug conjugates for cancer therapy. Annu. Rep. Med. Chem., 38, 229-237;Chari, R. V. (2008) Targeted cancer therapy: conferring specificity tocytotoxic drugs. Acc. Chem. Res. 41, 98-107]. More than 30 G-typeimmunoglobulins (IgG) and related agents have been approved over thepast 25 years mainly for cancers and inflammatory diseases.

An alternative strategy is to look to chemically conjugate smallanti-neoplastic molecules to mAbs, used both as carriers (increasedhalf-life) and as targeting agents (selectivity). Considerable efforthas been directed toward the use of monoclonal antibodies (mAbs) fortargeted drug delivery due to their high selectivities fortumor-associated antigens, favorable pharmacokinetics, and relativelylow intrinsic toxicities. The mAb-drug conjugates (ADCs) are formed bycovalently linking anticancer drugs to mAbs, usually through aconditionally stable linker system. Upon binding to cell surfaceantigens, mAbs used for most ADCs are actively transported to lysosomesor other intracellular compartments, where enzymes, low pH, or reducingagents facilitate drug release. There are, however, currently limitedADCs in development.

Antigens must have high tumor cell selectivity to limit toxicity andoff-target effects. A plethora of tumor-associated antigens have beeninvestigated in pre-clinical models and in clinical trials includingantigens over-expressed in B-cells (e.g., CD20, CD22, CD40, CD79),T-cells (CD25, CD30), carcinoma cells (HER2, EGFR, EpCAM, EphB2, PSMA),endothelial (endoglin), or stroma cells (fibroblast activated protein),to name a few [Teicher B A. Antibody-drug conjugate targets. Curr CancerDrug Targets 9(8):982-1004, 2009]. An important property for ADC targetsis their ability to be internalized; this can be an intrinsic feature ofthe antigen by itself, or it can be induced by the binding of theantibody to its antigen. Indeed, ADC internalization is crucial toreduce toxicity associated with an extracellular delivery of the drugpayload.

Regarding the conjugated small molecules and in contrast to the vastvariety of putative antigen targets, a limited number of families ofcytotoxic drugs used as payloads in ADCs are currently activelyinvestigated in clinical trials: calicheamycin (Pfizer), duocarmycins(Synthon), pyrrolobenzodiazepines (Spirogen), irinotecan (Immunomedics),maytansinoids (DM1 and DM4; ImmunoGen+Genentech/Roche, Sanofi-Aventis,Biogen Idec, Centocor/Johnson & Johnson, Millennium/Takeda), andauristatins (MMAE and MMAF; Seattle Genetics+Genentech/Roche,Medlmmune/AstraZeneca, Bayer-Schering, Celldex, Progenics, Genmab).Calicheamycin, duocarmycins and pyrrolobenzodiazepines are DNA minorgroove binders, irinotecan is a topoisomerase I inhibitor, whereasmaytansinoids and auristatins are tubulin depolymerization agents.

Interestingly, a representative of three of these cytotoxic-derived ADCshas reached late stage clinical trials. Trastuzumab emtansine (T-DM1),trastuzumab linked to a maytansinoid hemi-synthetic drug by a stablelinker (FDA approval on Feb. 22, 2013 for advanced HER2 positive breastcancer); Inotuzumab ozogamicin (CMC-544), a humanized anti-CD22 mAb(G5/44, IgG4) conjugated to calicheamycin with an acid labile linker(acetylphenoxy-butanoic) (B-cell non-Hodgkin's lymphoma); Brentuximabvedotin, a humanized anti-CD30 mAb linked to monomethyl auristatin E(MMAE), via a maleimidecaproyl-valyl-citrullinyl-p-aminobenzylcarbamatelinker (FDA approval on Aug. 19, 2011 for anaplastic large cell lymphomaand Hodking's lymphoma).

Linkers represent the key component of ADC structures. Several classesof second generation linkers have been investigated, includingacid-labile hydrazone linkers (lysosomes) (e.g. gemtuzumab andinotuzumab ozogamicin); disulfide-based linkers (reductive intracellularenvironment); non-cleavable thioether linkers (catabolic degradation inlysosomes) (e.g., trastuzumab emtansine); peptide linkers (e.g.citruline-valine) (lysosomal proteases like cathepsin-B) (e.g.brentuximab vedotin): see, for example, WO-A-2004/010957,WO-A-2006/060533 and WO-A-2007/024536. Purification of antibody-drugconjugates by size exclusion chromatography (SEC) has also beendescribed [see, e.g., Liu et al., Proc. Natl. Acad. Sci. USA, 93:8618-8623 (1996), and Chari et al., Cancer Research, 52: 127-131(1992)].

Trastuzumab (Herceptin) is a monoclonal antibody that interferes withthe HER2/neu receptor. Its main use is to treat certain breast cancers.The HER receptors are proteins that are embedded in the cell membraneand communicate molecular signals from outside the cell (moleculescalled EGFs) to inside the cell, and turn genes on and off. The HERproteins stimulate cell proliferation. In some cancers, notably certaintypes of breast cancer, HER2 is over-expressed, and causes cancer cellsto reproduce uncontrollably.

The HER2 gene is amplified in 20-30% of early-stage breast cancers,which makes it overexpress epidermal growth factor (EGF) receptors inthe cell membrane. In some types of cancer, HER2 may send signalswithout growth factors arriving and binding to the receptor, making itseffect in the cell constitutive; however, trastuzumab is not effectivein this case.

The HER2 pathway promotes cell growth and division when it isfunctioning normally; however when it is overexpressed, cell growthaccelerates beyond its normal limits. In some types of cancer thepathway is exploited to promote rapid cell growth and proliferation andhence tumor formation. In cancer cells the HER2 protein can be expressedup to 100 times more than in normal cells (2 million versus 20,000 percell). This overexpression leads to strong and constant proliferativesignaling and hence tumor formation. Overexpression of HER2 also causesdeactivation of checkpoints, allowing for even greater increases inproliferation.

In the compounds of the present invention, Ab is a moiety comprising atleast one antigen binding site. In an alternative embodiment, Ab can beany suitable agent that is capable of binding to a target cell,preferably an animal cell and more preferably, a human cell. Examples ofsuch agents include lymphokines, hormones, growth factors andnutrient-transport molecules (e.g. transferrin). In another example, Abmay be an aptamer, and may include a nucleic acid or a peptide aptamer.

Where Ab is a moiety comprising at least one antigen binding site, themoiety is preferably an antigen-binding peptide or polypeptide. In apreferred embodiment, the moiety is an antibody or an antigen-bindingfragment thereof.

The term ‘antibody’ in the drug conjugates of the present inventionrefers to any immunolglobulin, preferably a full-length immunoglobulin.Preferably, the term covers monoclonal antibodies, polyclonalantibodies, multispecific antibodies, such as bispecific antibodies, andantibody fragments thereof, so long as they exhibit the desiredbiological activity. Antibodies may be derived from any species, butpreferably are of rodent, for examples rat or mouse, human or rabbitorigin. Alternatively, the antibodies, preferably monoclonal antibodies,may be humanised, chimeric or antibody fragments thereof. The term‘chimeric antibodies’ may also include “primatised” antibodiescomprising variable domain antigen-binding sequences derived from anon-human primate (e.g., Old World Monkey, Ape etc) and human constantregion sequences. The immunoglobulins can also be of any type (e.g. IgG,IgE, IgM, IgD, and IgA), class (e.g., IgGI, IgG2, IgG3, IgG4, IgAI andIgA2) or subclass of immunoglobulin molecule.

The term ‘monoclonal antibody’ refers to a substantially homogenouspopulation of antibody molecules (i.e. the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts), produced by asingle clone of B lineage cells, often a hybridoma. Importantly, eachmonoclonal has the same antigenic specificity—i.e. it is directedagainst a single determinant on the antigen.

The production of monoclonal antibodies can be carried out by methodsknown in the art. However, as an example, the monoclonal antibodies canbe made by the hybridoma method (Kohler et al (1975) Nature 256:495),the human B cell hybridoma technique (Kozbor et al., 1983, ImmunologyToday 4: 72), or the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Alternatively, the monoclonal antibody can be produced usingrecombinant DNA methods (see, U.S. Pat. No. 4,816,567) or isolated fromphage antibody libraries using the techniques described in Clackson etal (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol.,222:581-597.

Polyclonal antibodies are antibodies directed against differentdeterminants (epitopes). This heterogenous population of antibody can bederived from the sera of immunised animals using various procedures wellknown in the art.

The term ‘bispecific antibody’ refers to an artificial antibody composedof two different monoclonal antibodies. They can be designed to bindeither to two adjacent epitopes on a single antigen, thereby increasingboth avidity and specificity, or bind two different antigens fornumerous applications, but particularly for recruitment of cytotoxic T-and natural killer (NK) cells or retargeting of toxins, radionuclides orcytotoxic drugs for cancer treatment (Holliger & Hudson, NatureBiotechnology, 2005, 23(9), 1126-1136). The bispecific antibody may havea hybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. Thisasymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation (WO94/04690; Suresh et al., Methods in Enzymology, 1986, 121:210; Rodrigueset al., 1993, J. of Immunology 151:6954-6961; Carter et al., 1992,Bio/Technology 10:163-167; Carter et al., 1995, J. of Hematotherapy4:463-470; Merchant et al., 1998, Nature Biotechnology 16:677-681.

Methods to prepare hybrid or bispecific antibodies are known in the art.In one method, bispecific antibodies can be produced by fusion of twohybridomas into a single ‘quadroma’ by chemical cross-linking or geneticfusion of two different Fab or scFv modules (Holliger & Hudson, NatureBiotechnology, 2005, 23(9), 1126-1136).

The term ‘chimeric’ antibody refers to an antibody in which differentportions are derived from different animal species. For example, achimeric antibody may derive the variable region from a mouse and theconstant region from a human. In contrast, a ‘humanised antibody’ comespredominantly from a human, even though it contains non-human portions.Specifically, humaised antibodies are human immunoglobulins (recipientantibody) in which residues from a hypervariable region of the recipientare replaced by residues from hypervariable regions of a non-humanspecies (donor antibody) such as mouse, rat, rabbit or nonhuman primatehaving the desired specificity, affinity and capacity. In someinstances, framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanisedantibodies may comprise residues that are not found in the recipientantibody or in the donor antibody. These modifications are made tofurther refine antibody performance. In general, the humanised antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the hypervariableloops correspond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The humanised antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin.

Recombinant antibodies such as chimeric and humanised monoclonalantibodies can be produced by recombinant DNA techniques known in theart. Completely human antibodies can be produced using transgenic micethat are incapable of expressing endogenous immunoglobulin heavy andlight chains genes, but which can express human heavy and light chaingenes. The transgenic mice are immunized in the normal fashion with aselected antigen. Monoclonal antibodies directed against the antigen canbe obtained using conventional hybridoma technology. The humanimmunoglobulin transgenes harboured by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93).

For a detailed discussion of this technology for producing humanantibodies and human monoclonal antibodies and protocols for producingsuch antibodies, see, for example, U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; 5,545,806; each of which is incorporated herein byreference in its entirety. Other human antibodies can be obtainedcommercially from, for example, Abgenix, Inc. (Freemont, Calif.) andGenpharm (San Jose, Calif.).

The term ‘antigen-binding fragment’ in the drug conjugates of thepresent invention refers to a portion of a full length antibody wheresuch antigen-binding fragments of antibodies retain the antigen-bindingfunction of a corresponding full-length antibody. The antigen-bindingfragment may comprise a portion of a variable region of an antibody,said portion comprising at least one, two, preferably three CDRsselected from CDR1, CDR2 and CDR3. The antigen-binding fragment may alsocomprise a portion of an immunoglobulin light and heavy chain. Examplesof antibody fragments include Fab, Fab′, F(ab′)₂, scFv, di-scFv, sdAb,and BiTE (Bi-specific T-cell engagers), Fv fragments includingnanobodies, diabodies, diabody-Fc fusions, triabodies and, tetrabodies;minibodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), and epitope-binding fragments of any of the abovethat immunospecifically bind to a target antigen such as a cancer cellantigens, viral antigens or microbial antigens, single-chain orsingle-domain antibody molecules including heavy chain only antibodies,for example, camelid VHH domains and shark V-NAR; and multispecificantibodies formed from antibody fragments. For comparison, a full lengthantibody, termed ‘antibody’ is one comprising a VL and VH domains, aswell as complete light and heavy chain constant domains.

The antibody may also have one or more effector functions, which referto the biological activities attributable to the Fc region (a nativesequence Fc region or amino acid sequence variant Fc region engineeredaccording to methods in the art to alter receptor binding) of anantibody. Examples of antibody effector functions include Clq binding;complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

The antibody can also be a functionally active fragment (also referredto herein as an immunologically active portion), derivative or analog ofan antibody that immunospecifically binds to a target antigen such as acancer cell antigen, viral antigen, or microbial antigen or otherantibodies bound to tumour cells. In this regard, functionally activemeans that the fragment, derivative or analog is able to elicitanti-idiotype antibodies that recognise the same antigen that theantibody from which the fragment, derivative or analog is derivedrecognised.

Specifically, in an exemplary embodiment the antigenicity of theidiotype of the immunoglobulin molecule can be enhanced by deletion offramework and CDR sequences that are C-terminal to the CDR sequence thatspecifically recognizes the antigen. To determine which CDR sequencesbind the antigen, synthetic peptides containing the CDR sequences can beused in binding assays with the antigen by any binding assay methodknown in the art (e.g., the BIA core assay), see, for example, Kabat etal., 1991, Sequences of Proteins of Immunological Interest, FifthEdition, National Institute of Health, Bethesda, Md; Kabat E et al.,1980, J. of Immunology 125(3):961-969).

The term ‘antibody’ may also include a fusion protein of an antibody, ora functionally active fragment thereof, for example in which theantibody is fused via a covalent bond (e.g., a peptide bond), at eitherthe N-terminus or the C-terminus to an amino acid sequence of anotherprotein (or portion thereof, such as at least 10, 20 or 50 amino acidportion of the protein) that is not the antibody. The antibody orfragment thereof may be covalently linked to the other protein at theN-terminus of the constant domain.

Furthermore, the antibody or antigen-binding fragments of the presentinvention may include analogs and derivatives of antibodies orantigen-binding fragments thereof that are either modified, such as bythe covalent attachment of any type of molecule as long as such covalentattachment permits the antibody to retain its antigen bindingimmunospecificity. Examples of modifications include glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular antibody unit or other protein, etc. Any of numerous chemicalmodifications can be carried out by known techniques, including, but notlimited to specific chemical cleavage, acetylation, formylation,metabolic synthesis in the presence of tunicamycin, etc. Additionally,the analog or derivative can contain one or more unnatural amino acids.

The antibodies or antigen-binding fragments of the present invention mayalso have modifications (e.g., substitutions, deletions or additions) inthe Fc domain of the antibody. Specifically, the modifications may be inthe Fc-hinge region and result in an increased binding for the FcRnreceptor (WO 97/34631).

In one embodiment, the antibody in the drug conjugate of the presentinvention may be any antibody or antigen-binding fragment thereof,preferably a monoclonal antibody that is useful in the treatment of adisease, preferably cancer. The cancer may be breast cancer, colorectalcancer, endometrial cancer, kidney cancer melanoma, leukaemias, lungcancer, multiple myeloma, lymphomas (e.g. Hodgkin's disease andnon-Hodgkin's Lymphoma), solid tumors such as sarcoma and carcinomas,melanoma, mesothelioma, osteosarcoma, ovarian cancer and renal cancer.In a preferred embodiment the cancer is lung cancer, colorectal cancer,breast cancer, pancreas carcinoma, kidney cancer, leukaemia, multiplemyeloma, lymphoma, gastric and ovarian cancer. In a more preferredembodiment the cancer is colorectal cancer, breast cancer, leukaemia,lymphoma, and ovarian cancer

Antibodies that may be useful in the treatment of cancer include, butare not limited to, antibodies against the following antigens: CA125(ovarian), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas),Lewis Y (carcinomas), Lewis X (carcinomas), alpha fetoprotein(carcinomas), CA 242 (colorectal), placental alkaline phosphatase(carcinomas), prostate specific antigen (prostate), prostatic acidphosphatase (prostate), epidermal growth factor (carcinomas) for exampleEGF receptor 2 protein (breast cancer), MAGE-I (carcinomas), MAGE-2(carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti-transferrinreceptor (carcinomas), p97 (melanoma), MUCl-KLH (breast cancer), CEA(colorectal), gplOO (melanoma), MARTI (melanoma), PSA (prostate), IL-2receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma),CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionicgonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma),mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogeneproduct (carcinomas). Some specific, useful antibodies include, but arenot limited to, BR96 mAb (Trail, P. A., et al Science (1993) 261,212-215), BR64 (Trail, P A, et al Cancer Research (1997) 57, 100-105,mAbs against the CD40 antigen, such as S2C6 mAb (Francisco, J. A., et alCancer Res. (2000) 60:3225-3231), mAbs against the CD70 antigen, such as1F6 mAb, and mAbs against the CD30 antigen, such as ACIO (Bowen, M. A.,et al (1993) J. Immunol., 151:5896-5906; Wahl et al., 2002 Cancer Res.62(13):3736-3742). Many other internalizing antibodies that bind totumor associated antigens can be used and have been reviewed (Franke, A.E., et al Cancer Biother Radiopharm. (2000) 15:459-476; Murray, J. L.,(2000) Semin Oncol, 27:64-70; Breitling, F., and Dubel, S., RecombinantAntibodies, John Wiley, and Sons, New York, 1998).

The present invention encompasses treating cancers associated with theseantibodies.

Other tumour-associated antigens include, but are not limited to,BMPR1B, E16, STEAPI, STEAP2, 0772P. MPF, Napi3b, Sema5b, PSCA hlg, ETBR,MSG783, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP, IL20Ra,Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79A, CXCR5, HLA-DOB,P2X5, CD72, LY64, FCRH1, IRTA2 and TENB2.

In an alternative embodiment, the antibody in the drug conjugate of thepresent invention may be an antibody or antigen-binding fragmentthereof, preferably a monoclonal antibody, that immunospecifically bindsto a viral antigen, microbial antigen or an antigen of a cell thatproduces autoimmune antibodies associated with autoimmune disease.

The viral antigen may include, but is not limited to, any viral peptide,polypeptide or protein such as HIV gpl20, HIV nef, RSV F glycoprotein,influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax,herpes simplex virus glycoprotein (e.g., Gb, Gc, Gd, and Ge) andhepatitis B surface antigen that is capable of eliciting an immuneresponse.

The microbial antigen may include, but is not limited to, any microbialpeptide, polypeptide, protein, saccharide, polysaccharide, or lipidmolecule (e.g., a bacterial, fungi, pathogenic protozoa, or yeastpolypeptide including, e.g., LPS and capsular polysaccharide) that iscapable of eliciting an immune response.

In a further embodiment, the antibody or antigen-binding fragment bindsto an epitope that is present on a cell, such as a tumour cell.Preferably, where the cell is a tumour cell, the tumour cell epitope isnot present on non-tumour cells, or is present at a lower concentrationor in a different steric configuration than in tumour cells.

In one embodiment, the antibody or antigen-binding fragment binds to anepitope present in the context of one of the following antigens: CA125,CA15-3, CA19-9 L6, Lewis Y, Lewis X, alpha fetoprotein, CA 242,placental alkaline phosphatase, prostate specific antigen, prostaticacid phosphatase, epidermal growth factor for example EGF receptor 2protein, MAGE-1, MAGE-2, MAGE-3, MAGE-4, anti-transferrin receptor, p97,MUCl-KLH, CEA, gplOO, MARTI, PSA, IL-2 receptor, CD20, CD52, CD33, CD22,human chorionic gonadotropin, CD38, CD40, mucin, P21, MPG, Neu oncogeneproduct, BMPR1B, E16, STEAPI, STEAP2, 0772P. MPF, Napi3b, Sema5b, PSCAhlg, ETBR, MSG783, TrpM4, CRIPTO, CD21, CD79b, FcRH2, HER2, NCA, MDP,IL20Ra, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79A, CXCR5,HLA-DOB, P2X5, CD72, LY64, FCRH1, IRTA2, TENB2, a viral antigen (such asany viral peptide, polypeptide or protein such as HIV gp120, HIV nef,RSV F glycoprotein, influenza virus neuraminidase, influenza virushemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., Gb,Gc, Gd, and Ge) and hepatitis B surface antigen) that is capable ofeliciting an immune response), microbial antigen (any microbial peptide,polypeptide, protein, saccharide, polysaccharide, or lipid molecule(e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptideincluding, e.g., LPS and capsular polysaccharide) that is capable ofeliciting an immune response) or an antigen of a cell that producesautoimmune antibodies associated with autoimmune disease.

In one embodiment, where the antigen is ErBB2 (also known as ERBB2,CD340 or HER2; such terms may be used interchangeably), the antibody orantigen-binding fragment may bind to one or more of the followingepitopes: ARHC L (SEQ ID NO: 1), QNGS (SEQ ID NO: 2) and PPFCVARC PSG(SEQ ID NO: 3). These epitopes correspond to positions 557-561, 570-573and 593-603 respectively of the human HER2 polypetide sequence(Accession: NM_004448, Version: NM_004448.3).

In another embodiment, the antibody may be any antibody known for thetreatment or prevention of viral or microbial infection—i.e. aninfectious disease. Examples of such antibodies include, but are notlimited to, PR0542 (Progenies) which is a CD4 fusion antibody useful forthe treatment of HIV infection; OsTAVIR (Protein Design Labs, Inc., CA)which is a human antibody useful for the treatment of hepatitis B virus;PROTOVIR. (Protein Design Labs, Inc., CA) which is a humanised IgG1antibody useful for the treatment of cytomegalovirus (CMV); and anti-LPSantibodies.

Other antibodies useful in the treatment of infectious diseases include,but are not limited to, antibodies against the antigens from pathogenicstrains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae,Neisseria gonorrheae, Neisseria meningitidis, Corynebacteriumdiphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridiumtetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiellaozaenas, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibriocolerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter(Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiellatarda, Yersinia enterocolitica, Yersinia pestis, Yersiniapseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigellasonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue,Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi,Leptospira icterohernorrhagiae, Mycobacterium tuberculosis, Pneumocystiscarinii, Francisella tularensis, Brucella abortus, Brucella suis,Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis,Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis,Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoebahistolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis,Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense,Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmaniabraziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodiumfalciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis,Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis,Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni,Schistosoma haematobium, and hookworms).

Other antibodies useful for the treatment of viral disease include, butare not limited to, antibodies against antigens of pathogenic viruses,including as examples and not by limitation: Poxviridae, Herpesviridae,Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae,Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae,Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles,respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae,Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus,Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae,Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus.

In an alternative embodiment, the antibody of the drug conjugate of thepresent invention may also be any antibody known for the treatment ofprevention of autoimmune disorders, such as, but not limited to,Th2-lymphocyte related disorders (e.g. atopic dermatitis, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, and graft versus host disease); Th1 lymphocyte-relateddisorders (e.g. rheumatoid arthritis, multiple sclerosis, psoriasis,Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primarybiliary cirrhosis, Wegener's granulomatosis, and tuberculosis);activated B lymphocyte-related disorders (e.g. systemic lupuserythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type Idiabetes); and Active Chronic Hepatitis, Addison's Disease, AllergicAlveolitis, Allergic Reaction, Allergic Rhinitis, Alport's Syndrome,Anaphlaxis, Ankylosing Spondylitis, Anti-phosholipid Syndrome,Arthritis, Ascariasis, Aspergillosis, Atopic Allergy, AtropicDermatitis, Atropic Rhinitis, Behcet's Disease, Bird-Fancier's Lung,Bronchial Asthma, Caplan's Syndrome, Cardiomyopathy, Celiac Disease,Chagas' Disease, Chronic Glomerulonephritis, Cogan's Syndrome, ColdAgglutinin Disease, Congenital Rubella Infection, CREST Syndrome,Crohn's Disease, Cryoglobulinemia, Cushing's Syndrome, Dermatomyositis,Discoid Lupus, Dresser's Syndrome, Eaton-Lambert Syndrome, EchovirusInfection, Encephalomyelitis, Endocrine opthalmopathy, Epstein-BarrVirus Infection, Equine Heaves, Erythematosis, Evan's Syndrome, Felty'sSyndrome, Fibromyalgia, Fuch's Cyclitis, Gastric Atrophy,Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis,Goodpasture's Syndrome, Graft v. Host Disease, Graves' Disease,Guillain-Barre Disease, Hashimoto's Thyroiditis, Hemolytic Anemia,Henoch-Schonlein Purpura, Idiopathic Adrenal Atrophy, IdiopathicPulmonary Fibritis, IgA Nephropathy, Inflammatory Bowel Diseases,Insulin-dependent Diabetes Mellitus, Juvenile Arthritis, JuvenileDiabetes Mellitus (Type I), Lambert-Eaton Syndrome, Laminitis, LichenPlanus, Lupoid Hepatitis, Lupus Lymphopenia, Meniere's Disease, MixedConnective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis,Pernicious Anemia, Polyglandular Syndromes, Presenile Dementia, PrimaryAgammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, PsoriaticArthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome,Schistosomiasis, Schmidt's Syndrome, Scleroderma, Shulman's Syndrome,Sjorgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthahnia, SystemicLupus Erythematosis, Takayasu's Arteritis, Temporal Arteritis,Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic EpidermalNecrolysis, Type B Insulin Resistance, Type I Diabetes Mellitus,Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom's Macroglobulemia andWegener's Granulomatosis.

Antibodies immunospecific for an antigen of a cell that is responsiblefor producing autoimmune antibodies can be obtained by any method knownto one of skill in the art such as, e.g., chemical synthesis orrecombinant expression techniques. Examples of autoimmune antibodiesinclude, but are not limited to, Anti-Nuclear Antibody; Anti ds DNA;Anti ss DNA, Anti Cardiolipin Antibody IgM, IgG; Anti PhospholipidAntibody IgM, IgG; Anti SM Antibody; Anti Mitochondrial Antibody;Thyroid Antibody; Microsomal Antibody; Thyroglobulin Antibody; AntiSCL-70; Anti-Jo; Anti-U1RNP; Anti-La/SSB; Anti SSA; Anti SSB; AntiPerital Cells Antibody; Anti Histones; Anti-RNP; C-ANCA; P-ANCA; Anticentromere; Anti-Fibrillarin, and Anti-GBM Antibody.

In another embodiment, the antibody of the drug conjugate of the presentinvention can be one that binds to both a receptor or a receptor complexexpressed on an activated lymphocyte, such as one associated with anautoimmune disease. The receptor or receptor complex can comprise animmunoglobulin gene superfamily member, a TNF receptor superfamilymember, an integrin, an interleukin, a cytokine receptor, a chemokinereceptor, a major histocompatibility protein, a lectin, or a complementcontrol protein. Non-limiting examples of suitable immunoglobulinsuperfamily members are CD2, CD3, CD4, CD5, CD8, CD13, CD19, CD22, CD28,CD79, CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting examples ofsuitable TNF receptor superfamily members are CD27, CD40, CD95/Fas,CD134/OX40, CD137/4-1BB, TNF-RI, TNFR-2, RANK, TACI, BCMA,osteoprotegerin, Apo2/TRAEL-RI, TRAIL-R2, TRAIL-R3, TRABL-R4, and APO-3.Non-limiting examples of suitable integrins are CDI la, CDIIb, CDIIc,CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, andCD104. Non-limiting examples of suitable lectins are C-type, S-type, andI-type lectin.

An antibody that binds a molecular target or an antigen of interest,e.g., ErbB2 antigen, is one capable of binding that antigen withsufficient affinity such that the antibody is useful in targeting a cellexpressing the antigen. Where the antibody is one which binds ErbB2, itwill usually preferentially bind ErbB2 as opposed to other ErbBreceptors, and may be one which does not significantly cross-react withother proteins such as EGFR, ErbB 3 or ErbB4. In such embodiments, theextent of binding of the antibody to these non-ErbB2 proteins (e.g.,cell surface binding to endogenous receptor) will be less than 10% asdetermined by fluorescence activated cell sorting (FACS) analysis orradioimmunoprecipitation (RIA). Sometimes, the anti-ErbB2 antibody willnot significantly cross-react with the rat neu protein, e.g., asdescribed in Schecter et al., Nature 312:513-516 (1984) and Drebin etal., Nature 312:545-548 (1984).

In another embodiment, the antibody of the drug conjugate or target ofthe present invention may be selected from an antibody or target in thebelow table. Such antibodies are immunospecific for a target antigen andcan be obtained commercially or produced by any method known in the artsuch as, e.g., recombinant expression techniques.

TABLE 1 Therapeutic monoclonal antibodies Name Trade name Target 3F8 GD2ganglioside 8H9 B7-H3 Abagovomab CA-125 (imitation) Abciximab ReoProCD41 7E3 Abituzumab CD51 Abrilumab Integrin α4β7 Actoxumab Clostridiumdifficile Adalimumab Humira TNF-α Adecatumumab EpCAM AtidortoxumabStaphylococcus aureus alpha toxin Aducanumab Beta-amyloid AfasevikumabIL17A and IL17F Afutuzumab CD20 Alemtuzumab Campath, Lemtrada CD52Alirocumab Praluent PCSK9 Altumomab Hybri-ceaker CEA AmatuximabMesothelin Andecaliximab gelatinase B Anetumab MSLN Anifrolumabinterferon α/β receptor Anrukinzumab IL-13 Apolizumab HLA-DR β-chainAprutumab FGFR2 Ascrinvacumab Activin receptor-like kinase 1 AselizumabL-selectin (CD62L) Atezolizumab Tecentriq PD-L1 AtidortoxumabStaphylococcus aureus alpha toxin Atinumab RTN4 Atorolimumab Rhesusfactor Avelumab Bavencio PD-L1 Azintuxizumab CD319 Bapineuzumab betaamyloid Basiliximab Simulect CD25 (α chain of IL-2 receptor) Bavituximabphosphatidylserine BCD-100 PD-1 Bectumomab LymphoScan CD22 BegelomabDPP4 Belantamab BCMA Belimumab Benlysta BAFF Bemarituzumab FGFR2Benralizumab Fasenra CD125 Berlimatoxumab Staphylococcus aureusbi-component leukocidin Bersanlimab ICAM-1 Bertilimumab CCL11(eotaxin-1) Besilesomab Scintimun CEA-related antigen BevacizumabAvastin VEGF-A Bezlotoxumab Zinplava Clostridium difficile BlinatomumabBlincyto CD19, CD3 Bimagrumab ACVR2B Bimekizumab IL 17A and IL17FBirtamimab Serum amyloid A protein Bivatuzumab CD44 v6 BIVV009 C1sBleselumab CD40 Blontuvetmab Biontress CD20 Blosozumab SOST BococizumabNeural apoptosis-regulated proteinase 1 Brazikumab IL23 BrentuximabAdcentris CD30 (TNFRSF8) Briakinumab IL-12, IL-23 Brodalumab Siliz IL-17Brontictuzumab Notch 1 Burosumab Crysvita FGF23 Cabiralizumab CSF1RCamidanlumab CD25 Camrelizumab Programmed cell death 1 Canakinumab HarisIL-1 Cantuzumab MUC-1 Capromab Prostascint prostatic carcinoma cellsCarlumab MCP-1 Carotuximab endoglin Catumaxomab Removab EpCAM, CD3 CC49TAG-72 CBR96 Lewis-Y antigen Cedelizumab CD4 Cemiplimab PCDC1Cergutuzumab IL2 Cetrelimab Programmed cell death 1 Cetuximab ErbituxEGFR Cibisatamab CEACAM5 Cixutumumab IGF-1 receptor (CD221) ClazakizumabIL6 Clenoliximab CD4 Clivatuzumab hPAM4-Cide MUC1 Codrituzumab glypican3 Cofetuzumab PTK7 Coltuximab CD19 Conatumumab TRAIL-R2 Concizumab TFPICosfroviximab ZMapp Ebolavirus glycoprotein CR6261 Influenza Ahemagglutinin Crenezumab 1-40-β-amyloid Crizanlizumab Selectin PCrotedumab GCGR Cusatuzumab CD70 Dacetuzumab CD40 Daclizumab ZenapaxCD25 (α chain of IL-2 receptor) Dalotuzumab IGF-1 receptor (CD221)Dapirolizumab CD154 (CD40L) pegol Daratumumab Darzalex CD38 DectrekumabIL-13 Demcizumab DLL4 Denintuzumab CD19 Denosumab Prolia RANKLDepatuxizumab EGFR Derlotuximab Histone complex Detumomab B-lymphomacell Dezamizumab Serum amyloid P component Dinutuximab Unituxin GD2ganglioside Diridavumab hemagglutinin Domagrozumab GDF-8 Drozitumab DR5Duligotuzumab ERBB3 (HER3) Dupilumab Dupixent IL4 Durvalumab ImfinziPD-L1 Dusigitumab ILGF2 Ecromeximab GD3 ganglioside Eculizumab SolirisC5 Edobacomab endotoxin Edrecolomab Panorex EpCAM Efalizumab RaptivaLFA-1 (CD11a) Eldelumab interferon gamma-induced protein Elezanumab RGMAElgemtumab ERBB3 (HER3) Elotuzumab Empliciti SLAMF7 Elsilimomab IL-6Emactuzumab CSF1R Emapalumab Gamifant Interferon gamma EmibetuzumabHHGFR Emicizumab Hemlibra Activated F9, F10 Enapotamab AXL EnavatuzumabTWEAK receptor Enfortumab nectin-4 Enlimomab pegol ICAM-1 (CD54)Enoblituzumab CD276 Enokizumab IL9 Enoticumab DLL4 Ensituximab 5ACEpitumomab episialin Epratuzumab CD22 Eptinezumab Calcitoningene-related peptide Erenumab Aimovig CGRP Ertumaxomab Rexomun HER2/neu,CD3 Etaracizumab Abegrin integrin α_(v)β₃ Etigilimab TIGIT Etrolizumabintegrin β₇ Evinacumab Angiopoietin 3 Evolocumab Repatha PCSK9Exbivirumab hepatitis B surface antigen Fanolesomab NeutroSpec CD15Faralimomab interferon receptor Faricimab VEGF-A and Ang-2 Farletuzumabfolate receptor 1 Fasinumab HNGF FBTA05 Lymphomun CD20 Felvizumabrespiratory syncytial virus Fezakinumab IL-22 Fibatuzumab Ephrinreceptor A3 Ficlatuzumab HGF Figitumumab IGF-1 receptor (CD221)Firivumab Influenza A virus hemagglutinin Flanvotumab TYRP1(glycoprotein 75) Fletikumab IL-20 Fontolizumab HuZAF IFN-γ ForalumabCD3 epsilon Foravirumab rabies virus glycoprotein FremanezumabCalcitonin gene-related peptide alpha Fresolimumab TGF-β Frunevetmab NGFFulranumab NGF Futuximab EGFR Galcanezumab calcitonin Galiximab CD80Ganitumab 1 receptor (CD221) Gantenerumab beta amyloid GatipotuzumabMUC1 Gavilimomab CD147 (basigin) Gedivumab Hemagglutinin HA GemtuzumabMylotarg CD33 Gevokizumab IL-1β Gilvetmab PCDC1 Gimsilumab CSF2Girentuximab Rencarex carbonic anhydrase 9 (CA-IX) Glembatumumab GPNMBGolimumab Simponi TNF-α Gomiliximab CD23 (IgE receptor) Gosuranemab tauprotein Guselkumab Tremfya IL23 Ianalumab BAFF-R Ibalizumab Trogarzo CD4Ibritumomab Zevalin CD20 Icrucumab VEGFR-1 Idarucizumab Praxbinddabigatran Ifabotuzumab EPHA3 Iladatuzumab CD97B IMAB362 CLDN18.2Imalumab MIF Imaprelimab MCAM Imciromab Myoscint cardiac myosinImgatuzumab EGFR Inclacumab selectin P Indatuximab SDC1 indusatumabGUCY2C inebilizumab CD19 Infliximab Remicade TNF-α Inolimomab CD25 (αchain of IL-2 receptor) Inotuzumab Besponsa CD22 Intetumumab CD51Ipilimumab Yervoy CD152 Iomab-B CD45 Iratumumab CD30 (TNFRSF8)Isatuximab CD38 Iscalimab CD40 Istiratumab IGF1R, CD221 ItolizumabAlzumab CD6 Ixekizumab Taltz IL-17A Keliximab CD4 Labetuzumab CEA-CideCEA Lacnotuzumab CSF1, MCSF Ladiratuzumab LIV-1 Lanadelumab kallikreinLandogrozumab GDF-8 Laprituximab EGFR Larcaviximab Ebolavirusglycoprotein Lebrikizumab IL-13 Lemalesomab NCA-90 (granulocyte antigen)Lendalizumab C5 Lenvervimab Hepatitis B surface antigen Lenzilumab CSF2Lerdelimumab TGF beta 2 Leronlimab CCR5 Lesofavumab Hemagglutinin HALexatumumab TRAIL-R2 Libivirumab hepatitis B surface antigenLifastuzumab Phosphate-sodium co-transporter Ligelizumab IGHE LilotomabCD37 Lintuzumab CD33 Lirilumab KIR2D Lodelcizumab PCSK9 LokivetmabCytopoint Canis lupus familiaris IL31 Loncastuximab CD19 LosatuxizumabEGFR, ERBB1 HER1 Lorvotuzumab CD56 Lucatumumab CD40 Lulizumab pegol CD28Lumiliximab CD23 (IgE receptor) Lumretuzumab ERBB3 (HER3) LupartumabLYPD3 Lutikizumab Interleukin 1 alpha MABp1 Xilonix IL1A MapatumumabTRAIL-R1 Margetuximab HER2 Marstacimab TFPI Maslimomab T-cell receptorMavrilimumab GMCSF receptor α-chain Matuzumab EGFR Mepolizumab BosatriaIL-5 Metelimumab TGF beta 1 Milatuzumab CD74 Minretumomab TAG-72Mirikizumab IL23A Mirvetuximab Folate receptor alpha Mitumomab GD3ganglioside Modotuximab EGFR extracellular domain III MogamulizumabPoteligeo CCR4 Monalizumab NKG2A Morolimumab Rhesus factor MosunetuzumabCD3E, MS4A1, CD20 Motavizumab Numax respiratory syncytial virusMoxetumomab CD22 Muromonab-CD3 Orthoclone OKT3 CD3 Namilumab CSF2Naratuximab CD37 Narnatumab RON Natalizumab Tysabri integrin α₄Navicixizumab DLL4 Navivumab Influenza A virus hemagglutinin HANaxitamab C-Met Nebacumab endotoxin Necitumumab Portrazza EGFRNemolizumab IL31RA Nerelimomab TNF-α Nesvacumab angiopoietin 2 NetakimabInterleukin 17A Nimotuzumab Theracim, Theraloc EGFR Nirsevimab RSVFRNivolumab Opdivo PD-1 Obiltoxaximab Anthim Bacillus anthracis anthraxObinutuzumab Gazyva CD20 Ocaratuzumab CD20 Ocrelizumab Ocrevus CD20Odulimomab LFA-1 (CD11a) Ofatumumab Arzerra CD20 Olaratumab LartruvoPDGF-R α Oleclumab 5′-nucleotidase Olendalizumab Complement C5aOlokizumab IL6 Omalizumab Xolair IgE Fc region OMS721 MASP-2 Onartuzumabhuman scatter factor receptor kinase Ontuxizumab TEM1 Onvatilimab VSIROpicinumab LINGO-1 Oregovomab OvaRex CA-125 Orticumab oxLDL OtelixizumabCD3 Otilimab GMCSF Otlertuzumab CD37 Oxelumab OX-40 Ozanezumab NOGO-AOzoralizumab TNF-α Pagibaximab lipoteichoic acid Palivizumab Synagis, Fprotein of respiratory syncytial virus Abbosynagis Pamrevlumab CTGFPanitumumab Vectibix EGFR Pankomab Tumor specific glycosylation of MUC1Panobacumab Pseudomonas aeruginosa Parsatuzumab EGFL7 Pascolizumab IL-4Pasotuxizumab Folate hydrolase Pateclizumab LTA Patritumab ERBB3 (HER3)Pembrolizumab Keytruda PD1 Pemtumomab Theragyn MUC1 Perakizumab IL17APertuzumab Omnitarg HER2/neu Pidilizumab PD-1 Pinatuzumab CD22Pintumomab adenocarcinoma antigen Placulumab human TNF Plozalizumab CCR2Pogalizumab TNFR superfamily member 4 Polatuzumab CD79B Ponezumab humanbeta-amyloid Porgaviximab Zaire evolavirus glycoprotein PrasinezumabNACP Prezalizumab ICOSL Priliximab CD4 Pritoxaximab E. coli shiga toxintype-1 Pritumumab vimentin PRO 140 CCR5 Quilizumab IGHE RacotumomabVaxira NGNA ganglioside Radretumab fibronectin extra domain-BRafivirumab rabies virus glycoprotein Ralpancizumab Neuralapoptosis-regulated proteinase 1 Ramucirumab Cyramza VEGFR2 RanevetmabNGF Ravagalimab CD40 Ravulizumab C5 Raxibacumab anthrax toxin,protective antigen Refanezumab Myelin-associated glycoproteinRegavirumab cytomegalovirus glycoprotein B Relatlimab LAG3 RemtolumabInterleukin 17 alpha, TNF Reslizumab Cinqair IL-5 Rilotumumab HGFRinucumab Platelet-derived growth factor receptor beta. RisankizumabIL23A Rituximab MabThera, Rituxan CD20 Rivabazumab pegol Pseudomonasaeruginosa type III secretion system Robatumumab IGF-1 receptor (CD221)Rmab RabiShield Rabies virus G glycoprotein Roledumab RHD RomilkimabInterleukin 13 Romosozumab Evenity sclerostin Rontalizumab IFN-αRosmantuzumab Root plate-specific spondin 3 Rovalpituzumab DLL3Rovelizumab LeukArrest CD11, CD18 Rozanolixizumab FCGRT RuplizumabAntova CD154 (CD40L) SA237 IL-6R Sacituzumab TROP-2 Samalizumab CD200Samrotamab LRRC15 Sapelizumab IL6R Sarilumab Kevzara IL6 SatralizumabIL6 receptor Satumomab TAG-72 Secukinumab Cosentyx IL-17A SelicrelumabCD40 Seribantumab ERBB3 (HER3) Setoxaximab E. coli shiga toxin type-2Setrusumab SOST Sevirumab cytomegalovirus Sibrotuzumab FAP SGN-CD19ACD19 SHP647 Mucosal addressin cell adhesion molecule Sifalimumab IFN-αSiltuximab Sylvant IL-6 Simtuzumab LOXL2 Sintilimab PD-1 Siplizumab CD2Sirtratumab SLITRK6 Sirukumab IL-6 Sofituzumab CA-125 Solanezumab betaamyloid Sonepcizumab sphingosine-1-phosphate Sontuzumab episialinSpartalizumab PDCD1, CD279 Stamulumab myostatin Suptavumab RSVFRSutimlimab C1S Suvizumab HIV-1 Suvratoxumab Staphylococcus aureus alphatoxin Tabalumab BAFF Tacatuzumab AFP-Cide alpha-fetoproteinTalacotuzumab CD123 Talizumab IgE Tamtuvetmab Tactress CD52 TanezumabNGF Taplitumomab CD19 Tarextumab Notch receptor Tavolimab CD134Tefibazumab Aurexis clumping factor A Telisotuzumab HGFR Tenatumomabtenascin C Teneliximab CD40 Teplizumab CD3 Tepoditamab Dendriticcell-associated lectin 2 Teprotumumab IGF-1 receptor (CD221) TesidolumabC5 Tetulomab CD37 Tezepelumab TSLP Tibulizumab BAFF Tildrakizumab IlumyaIL23 Tigatuzumab TRAIL-R2 Timigutuzumab HER2 Timolumab AOC3 TiragotumabTIGIT Tislelizumab PCDC1, CD279 Tisotumab Coagulation factor IIITocilizumab Actemra, RoActemra IL-6 receptor Tomuzotuximab EGFR, HER1Toralizumab CD154 (CD40L) Tosatoxumab Staphylococcus aureus TositumomabBexxar CD20 Tovetumab CD140a Tralokinumab IL-13 Trastuzumab HerceptinHER2/neu TRBS07 Ektomab GD2 ganglioside Tregalizumab CD4 TremelimumabCTLA-4 Trevogrumab Growth differentiation factor 8 Tucotuzumab EpCAMTuvirumab hepatitis B virus Ublituximab MS4A1 Ulocuplumab CXCR4 (CD184)Urelumab 4-1BB (CD137) Urtoxazumab Escherichia coli Ustekinumab StelaraIL-12, IL-23 Utomilumab 4-1BB (CD137) Vadastuximab CD33 Vanalimab CD40Vandortuzumab STEAP1 Vantictumab Frizzled receptor Vanucizumabangiopoietin 2 Vapaliximab AOC3 (VAP-1) Varisacumab VEGF-A VarlilumabCD27 Vatelizumab ITGA2 (CD49b) Vedolizumab Entyvio integrin α₄β₇Veltuzumab CD20 Vepalimomab AOC3 (VAP-1) Vesencumab NRP1 VisilizumabNuvion CD3 Volociximab integrin α₅β₁ Vonlerolizumab CD134 VopratelimabICOS Vorsetuzumab CD70 Votumumab HumaSPECT tumor antigen CTAA16.88Vunakizumab Interleukin 17 alpha Xentuzumab IGF1, IGF2 XMAB-5574 CD19Zalutumumab HuMax-EGFr EGFR Zanolimumab HuMax-CD4 CD4 Zatuximab HER1Zenocutuzumab ERBB3, HER3 Ziralimumab CD147 (basigin) ZolbetuximabCLDN18 Zolimomab CD5

In addition to the above, the antibody of the drug antibody conjugate ofthe present invention may be Vitaxin which is a humanised antibody forthe treatment of sarcoma; Smart IDIO which is a humanised anti-HLA-DRantibody for the treatment of non-Hodgkin's lymphoma; Oncolym which is aradiolabeled murine anti-HLA-DrIO antibody for the treatment ofnon-Hodgkin's lymphoma; and Allomune which is a humanised anti-RD2 mAbfor the treatment of Hodgkin's Disease or non-Hodgkin's lymphoma.

The antibody of the drug conjugate of the present invention may also beany antibody-fragment known for the treatment of any disease, preferablycancer. Again, such antibody fragments are immunospecific for a targetantigen and can be obtained commercially or produced by any method knownin the art such as, e.g., recombinant expression techniques. Examples ofsuch antibodies available include any from the below table.

TABLE 2 Therapeutic monoclonal antibody fragments Fragment type/formatName Trade name Target Fab/chimeric abciximab ReoPro CD41 (integrinalpha-IIb) Fab/humanised abrezekimab Interleukin 13 F(ab′)₂/mouseAfelimomab TNF-α F(ab′)₂/humanised Alacizumab VEGFR2 pegol Fab/mouseAnatumomab TAG-72 Fab/ovine CroFab Snake venom Fab/ovine DigiFab DigoxinFab/ovine Digibind Digoxin Fab′/mouse arcitumomab CEA-scan CEAFab′/mouse bectumomab LymphoScan CD22 Fab′/mouse biciromab FibriScintfibrin II, beta chain BiTE/mouse Blinatumomab Blincyto CD19scFv/humanised brolucizumab VEGFA sdAb/humanised caplacizumab CabliviVWF Fab′/PEGylated certolizumab Cimzia TNF-α humanised pegolFab/humanised citatuzumab EpCAM F(ab′)₂/mouse dorlimomab unknownscFv/chimeric duvortuxizumab CD19, CD3E humanised scFv/human efungumabMycograb Hsp90 F(ab′)₂/humanised erlizumab ITGB2 (CD18) Di-scFyflotetuzumab IL-3 receptor scFv/human gancotamab unknown F(ab′)₂/mouseigovomab Indimacis-125 CA-125 Fab/humanised lampalizumab CFDscFv/humanised letolizumab TRAP Fab/mouse nacolomab C242 antigenFab/mouse naptumomab 5T4 Fab/mouse nofetumomab unknown scFv/humanisedoportuzumab Vicinium EpCAM Fab/humanised ranibizumab Lucentis VEGF-ABiTE/mouse Solitomab EpCAM Fab′/mouse sulesomab LeukoScan NCA-90(granulocyte antigen) Fab Tadocizumab integrin α_(IIb)β₃ Fab/mouseTelimomab unknown scFv/humanised Vobarilizumab IL6R Fab/humanisedThromboview D-dimer Fab/PEGylated CDP791 VEGF humanised Fab/bispecificMDX-H210 Her2/Neu & humanised CD64 (γFcR1) scFv/humanised PexelizumabComplement C5 (ScFv)₄ fused to CC49 TAG-72 streptavidin mousePancarcinoma antigen ScFv fused to β- SGN-17 P97 antigen lactamase humanScFv fused to PEG F5 scFv-PEG Her2 human Immunoliposome Diabody C6.5K-AHer2/Neu (V_(H)-V_(L))2 human Diabody L19 EDB domain of (V_(H)-V_(L))2L19-yIFN fibronectin human Diabody T84.66 CEA (V_(L)-V_(H))2 humanMinibody T84.66 CEA (scF_(v)-C_(H)3)₂ murine-human chimera (minibody)Minibody 10H8 Her2 murine-human chimera (minibody) S_(c)F_(v) dimer FcT84.66 CEA (S_(c)Fv)₂-FC murine-human chimera (minibody) Bispecific scFvr28M CD28 and MAP (V_(L)-V_(H)-V_(H)-V_(L)) mouse Bispecific scFv BiTEMT103 CD19 and CD3 (V_(L)-V_(H)-V_(H)-V_(L)) origin unknown BispecificscFv BiTE Ep-CAM and (V_(L)-V_(H)-V_(H)-V_(L)) CD3 origin unknownBispecific tandem diabody Tandab CD19 & CD3 (VH-VL- VH -VL) (mouse)VhH-β-lactamase fusion Nanobody CEA camelid Dab/human Anti-TNFα dAb TNFαVhH/camelid Nanobody TNFα VhH/camelid Nanobody Von Willebrand factor Fabfragment, antigen-binding (one arm) F(ab′)2 fragment, antigen-binding,including hinge region (both arms) Fab′fragment, antigen-binding,including hinge region (one arm) scFv single-chain variable fragmentdi-scFv dimeric, single-chain variable fragment

(Holliger & Hudson, Nature Biotechnology, 2005, 23(9), 1126-1136).

In a preferred embodiment, the antibody in the drug conjugates of thepresent invention targets a cell surface antigen.

In preferred embodiments, the antibody in the drug conjugates of thepresent invention may bind to a receptor encoded by the ErbB gene. Theantibody may bind specifically to an ErbB receptor selected from EGFR,HER2, HER3 and HER4. Preferably, the antibody in the drug conjugate mayspecifically bind to the extracellular domain of the HER2 receptor andinhibit the growth of tumour cells which overexpress the HER2 receptor.The antibody of the drug conjugate may be a monoclonal antibody, e.g. amurine monoclonal antibody, a chimeric antibody, or a humanisedantibody. Preferably, the humanised antibody may be huMAb4D5-1,huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7or huMAb4D5-8 (Trastuzumab), particularly preferably Trastuzumab. Theantibody may also be an antibody fragment, e.g. a Fab fragment.

Other preferred antibodies include:

(i) anti-CD4 antibodies. The antibody of the drug conjugate may be amonoclonal antibody, e.g. a murine monoclonal antibody, a chimericantibody, or a humanised antibody;(ii) anti-CD5 antibodies. The antibody of the drug conjugate may be amonoclonal antibody, e.g. a murine monoclonal antibody, a chimericantibody, or a humanised antibody;(iii) anti-CD13 antibodies. The antibody of the drug conjugate may be amonoclonal antibody, e.g. a murine monoclonal antibody, a chimericantibody, or a humanised antibody;(iv) anti-CD20 antibodies. The antibody of the drug conjugate may be amonoclonal antibody, e.g. a murine monoclonal antibody, a chimericantibody, or a humanised antibody. Preferably, the humanised antibody isRituximab or an antibody fragment thereof, e.g. a Fab fragment; and(v) anti-CD30 antibodies. The antibody of the drug conjugate may be amonoclonal antibody, e.g. a murine monoclonal antibody, a chimericantibody, or a humanised antibody. Preferably the humanised antibody isBrentuximab vedotin or an antibody fragment thereof.

In one embodiment of the invention, the drug antibody conjugate maydemonstrate one or more of the following: (i) increased cytotoxicity (ora decrease in cell survival), (ii) increased cytostatic activity(cytostasis), (iii) increased binding affinity to the target antigen orepitope, (iv) increased internalisation of the conjugate, (v) reductionof patient side effects, and/or (vi) improved toxicity profile. Suchincrease may be relative to a known drug antibody conjugate in the artthat binds the same or a different epitope or antigen.

Processes For The Preparation Of The Drug Antibody Conjugates

The drug antibody conjugates of the present invention can be preparedaccording to techniques that are well known in the art. Processes forconjugating moieties comprising at least one antigen binding siteantibodies such as antibodies to a number of different drugs usingdifferent processes have been described and exemplified previously in,for example, WO-A-2004/010957, WO-A-2006/060533 and WO-A-2007/024536,the contents of which are incorporated herein by reference thereto.These involve use of a linker group that derivatises the drug, toxin orradionuclide in such a way that it can then be attached to the moietysuch as an antibody. Attachment to the moiety such as an antibody istypically by one of three routes: via free thiol groups in cysteinesafter partial reduction of disulfide groups in the antibody; via freeamino groups in lysines in the antibody; and via free hydroxyl groups inserines and/or threonines in the antibody. The attachment method variesdepending upon the site of attachment on the moiety such as an antibody.Purification of antibody-drug conjugates by size exclusionchromatography (SEC) has also been described [see, e.g., Liu et al.,Proc. Natl. Acad. Set (USA), 93: 8618-8623 (1996), and Chari et al.,Cancer Research, 52: 127-131 (1992)].

As previously noted, the drug payloads of the drug conjugates of thepresent invention are ecteinascidin derivatives disclosed in, or fallwithin the scope of, International patent application no.PCT/EP2018/060868, the contents of which are incorporated herein byreference thereto. These compounds are synthesised according to theprocesses described in the present application.

As noted earlier, there is provided a process for the preparation of adrug conjugate according to the present invention comprising conjugatinga moiety Ab comprising at least one antigen binding site and a drug D offormula (IH), (IHa) or (IHb), Ab and D being as defined herein.

One example of a process for the preparation of a drug conjugate of thepresent invention involves the preparation of drug antibody conjugatesof formula (G) or (G′) of the present invention as follows:

said process comprising the following steps:(i) reacting a drug (D-H) of formula (IH)-H:

wherein the substituents in the definitions of (IH)-H are as definedabove for formula (IH), with a compound of formula (D′) or (E):

to give a compound of formula (F) or (F′), respectively:

(ii) partial reduction of one or more disulfide bonds in the antibody tobe conjugated to give a reduced antibody Ab-SH having free thiol groups:

and(iii) reaction of the partially reduced antibody Ab-SH having free thiolgroups with the compound of formula (F) or (F′) produced in step (i) togive the desired drug antibody conjugate of formula (G) or (G′)respectively:

In another preferred embodiment of this process, the antibody isselected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab,Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13antibody and an anti-CD 30 antibody, or an antigen-binding fragment oran immunologically active portion thereof, or it is selected fromTrastuzumab and anti-CD13 antibody or an antigen-binding fragment or animmunologically active portion thereof, and most preferably it isTrastuzumab or an antigen-binding fragment or an immunologically activeportion thereof. Furthermore, the partial reduction of this monoclonalantibodody is performed using tris[2-carboxyethyl]phosphinehydrochloride (TCEP).

Another example of a process for the preparation of a drug conjugate ofthe present invention involves the preparation of drug antibodyconjugates of formula (W) or (W′) of the present invention as follows:

said process comprising the following steps:(i) reacting the antibody with 2-iminothiolane hydrochloride (Traut'sreagent) to give a thiol-activated antibody:

(ii) reacting the thiol-activated antibody with the compound of formula(F) or (F′), to give the desired drug antibody conjugate of formula (W)or (W′), respectively.

In another preferred embodiment of this process, the antibody isselected from Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab,Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13antibody and an anti-CD 30 antibody, or an antigen-binding fragment oran immunologically active portion thereof, or it is selected fromTrastuzumab and anti-CD13 antibody or an antigen-binding fragment or animmunologically active portion thereof, and most preferably it isTrastuzumab or an antigen-binding fragment or an immunologically activeportion thereof.

Another example of a process for the preparation of a drug antibodyconjugate of the present invention, involves the preparation of drugantibody conjugates of formula (O) or (P) as follows:

said process comprising the following steps:(i) either:(a) reacting a drug (D-H) of formula (IH)-H:

wherein the substituents in the definitions of (IH)-H are as definedabove, with a compound of formula X₂—C(O)-X₁ wherein X1 and X2 areleaving groups to give a compound of formula (B):

and the point of attachment of the —(C═O)X₁ moiety is the free —NH₂group of the compound of formula D-H, or(b) reacting said drug (D-H) of formula (IH)-H as defined above with4-nitro-phenylchloroformate to give a compound of formula (J):

and the point of attachment of the (4-nitrophenyl)-O—CO— group is thesame as that for the X1(CO) moiety in (a) above;(ii) either:

(c) reacting the compound of formula (B) produced in step (i) with ahydroxy compound of formula HO—(CH₂)₁₋₆NHProt^(NH) and removing theProt^(NH) group from the coupled compound to give a compound of formula(C):

and then reacting the resulting compound of formula (C) with a compoundof formula Me-S—S—(CH₂)₁₋₃—CO₂H to give a compound of formula (K):

(d) reacting the compound (J) produced in step (i) with a compound offormula HO—(CH₂)₁₋₃SProt^(SH) and removing the Prot^(SH) group from thecoupled compound to give a compound of formula (L):

(iii) reacting (K) or (L) produced in step (ii) with dithiothreitolunder disulfide reducing conditions to give compounds of formula (M) and(N) respectively:

(iv) reacting the antibody to be conjugated withsuccininimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate toderivatise said antibody at one or more lysine groups with asuccininimidyl-4-(N-maleimidomethyl)cyclohexane-1-carbonyl group:

(v) reacting the derivatised antibody produced in step (iv) with either(M) or (N) produced in step (iii) to give the desired drug antibodyconjugate of formula (O) or (P):

The compound of formula X₂—C(O)-X₁ is preferably1,1′-carbonyldiimidazole. Similarly, the hydroxy compound reacted withthe compound of formula (B) is preferably HO—(CH₂)₂₋₄-NHProtNH, and morepreferably HO—(CH₂)₃-NHProt^(NH).In one preferred embodiment of this invention, the compound reacted withthe compound of formula (C) to give the compound of formula (K) is3-(methyldisulfanyl)propanoic acid.In another preferred embodiment, the compound HO—(CH₂)₁₋₃SProt^(SH) thatis reacted with a compound of formula (J) to give a compound of formula(L) is HO—(CH₂)₃SProt^(SH).

Where attachment to the drug linker moiety is via free thiol groups incysteines after partial reduction of disulfide groups in the moietycomprising at least one antigen binding site such as a monoclonalantibody, the partial reduction is typically conducted by first dilutingto a suitable concentration and buffering the solution before partialreduction of the disulfide bonds by means of the addition of a suitablereducing agent such as tris[2-carboxyethyl]phosphine hydrochloride(TCEP) or dithiothreitol (DTT). By choosing appropriate ratios of themoiety to be reduced such as a monoclonal antibody and the reducingagent, the reaction conditions and the time of the reduction it ispossible to obtain a desired free thiol to moiety ratio, e.g. four freethiol groups per monoclonal antibody.

The partially reduced moiety such as the partially reduced monoclonalantibody having the free thiol groups, prepared as described above, isthen reacted with drug-linker compounds of the invention of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-L1 (wherein the group L₁ in such compound isa maleimide group which is free to react with the thiol groups). Theresulting drug antibody conjugates are purified by any suitable meansknown in the art, e.g. by size exclusion chromatography (SEC) [see,e.g., Liu et al., Proc. Natl. Acad. Sci. USA, 93: 8618-8623 (1996), andChari et al., Cancer Research, 52: 127-131 (1992)].

In one preferred embodiment of this invention, the partially reducedmonoclonal antibody is Trastuzumab or an anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof,preferably Trastuzumab or an antigen-binding fragment or animmunologically active portion thereof; or preferably an anti-CD13antibody or an antigen-binding fragment or an immunologically activeportion thereof.

In an alternative embodiment of the invention, lysines in the moietycomprising at least one antigen binding site such as a monoclonalantibody can first be reacted withsuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. A freeamine group on an antibody can react with the N-hydroxysuccinimide esterto give a maleimide-activated antibody:

The maleimide-activated antibody can then be reacted with a compound offormula D-(X)_(b)-(AA)_(w)-(T)_(g)-H having a reactive thiol moiety.

In an alternative embodiment of the invention, lysines in the moietycomprising at least one antigen binding site such as a monoclonalantibody can first be reacted with 2-iminothiolane hydrochloride(Traut's reagent). A free amine group on an antibody can react with theimidic thiolactone to give a thiol-activated antibody.

One specific example of processes for the preparation of drug antibodyconjugates of formula [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]n-Ab of thepresent invention by conjugation via free thiol groups in cysteinesafter partial reduction of disulfide groups in the antibody is shown inFIG. 1 .

Another specific example of processes for the preparation of drugantibody conjugates of formula [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]n-Ab ofthe present invention by conjugation with free amino groups in lysinesafter reaction of the antibody with Traut's reagent is shown in FIG. 2 .

Compositions Comprising the Drug Antibody Conjugate of the Invention andUses Thereof

There is also provided a pharmaceutical composition comprising a drugconjugate according to the present invention and a pharmaceuticallyacceptable carrier. Examples of the administration form of a drugconjugate having the general formula[D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]n-Ab of the present invention includewithout limitation oral, topical, parenteral, sublingual, rectal,vaginal, ocular, and intranasal. Parenteral administration includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques. Preferably, the compositions areadministered parenterally. Pharmaceutical compositions of the inventioncan be formulated so as to allow a drug conjugate of the presentinvention to be bioavailable upon administration of the composition toan animal, preferably human. Compositions can take the form of one ormore dosage units, where for example, a tablet can be a single dosageunit, and a container of a drug antibody conjugate of the presentinvention in aerosol form can hold a plurality of dosage units.

The pharmaceutically acceptable carrier or vehicle can be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid. In addition, the carrier(s) can begaseous, so as to provide an aerosol composition useful in, for example,inhalatory administration. The term “carrier” refers to a diluent,adjuvant or excipient, with which a drug antibody conjugate of thepresent invention is administered. Such pharmaceutical carriers can beliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. The carriers can be saline, gum acacia,gelatin, starch paste, talc, keratin, colloidal silica, urea, and thelike. In addition, auxiliary, stabilizing, thickening, lubricating andcoloring agents can be used. In one embodiment, when administered to ananimal, the drug antibody conjugates of the present invention orcompositions and pharmaceutically acceptable carriers are sterile. Wateris a preferred carrier when the drug antibody conjugates of the presentinvention are administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceutical carriersalso include excipients such as starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The presentcompositions, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents.

When intended for oral administration, the composition is preferably insolid or liquid form, where semi-solid, semi-liquid, suspension and gelforms are included within the forms considered herein as either solid orliquid.

As a solid composition for oral administration, the composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such asstarch, lactose or dextrins, disintegrating agents such as alginic acid,sodium alginate, corn starch and the like; lubricants such as magnesiumstearate; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin; a flavoring agent such as peppermint,methyl salicylate or orange flavoring; and a coloring agent.

When the composition is in the form of a capsule (e.g. a gelatincapsule), it can contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition can be in the form of a liquid, e.g. an elixir, syrup,solution, emulsion or suspension. The liquid can be useful for oraladministration or for delivery by injection. When intended for oraladministration, a composition can comprise one or more of a sweeteningagent, preservatives, dye/colorant and flavor enhancer. In a compositionfor administration by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent can also be included.

The preferred route of administration is parenteral administrationincluding, but not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural,intranasal, intracerebral, intraventricular, intrathecal, intravaginalor transdermal. The preferred mode of administration is left to thediscretion of the practitioner, and will depend in part upon the site ofthe medical condition (such as the site of cancer). In a more preferredembodiment, the present drug antibody conjugates of the presentinvention are administered intravenously.

The liquid compositions of the invention, whether they are solutions,suspensions or other like form, can also include one or more of thefollowing: sterile diluents such as water for injection, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils such as synthetic mono or digylcerides,polyethylene glycols, glycerin, or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; and agents for the adjustmentof tonicity such as sodium chloride or dextrose. A parenteralcomposition can be enclosed in an ampoule, a disposable syringe or amultiple-dose vial made of glass, plastic or other material.Physiological saline is a preferred adjuvant.

The amount of the drug conjugate of the present invention that iseffective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro or in vivo assayscan optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the compositions will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances.

The compositions comprise an effective amount of a drug conjugate of thepresent invention such that a suitable dosage will be obtained. Thecorrect dosage of the compounds will vary according to the particularformulation, the mode of application, and its particular site, host andthe disease being treated, e.g. cancer and, if so, what type of tumor.Other factors like age, body weight, sex, diet, time of administration,rate of excretion, condition of the host, drug combinations, reactionsensitivities and severity of the disease shall be taken into account.Administration can be carried out continuously or periodically withinthe maximum tolerated dose.

The drug conjugate of the present invention or compositions can beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings.

In specific embodiments, it can be desirable to administer one or moredrug conjugates of the present invention or compositions locally to thearea in need of treatment. In one embodiment, administration can be bydirect injection at the site (or former site) of a cancer, tumor orneoplastic or pre-neoplastic tissue. In another embodiment,administration can be by direct injection at the site (or former site)of a manifestation of an autoimmune disease.

Pulmonary administration can also be employed, e.g. by use of an inhaleror nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the drug antibody conjugate of the presentinvention or compositions can be formulated as a suppository, withtraditional binders and carriers such as triglycerides.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained—release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse. Other examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

The pharmaceutical compositions can be prepared using methodology wellknown in the pharmaceutical art. For example, a composition intended tobe administered by injection can be prepared by combining a drugconjugate of the present invention with water so as to form a solution.A surfactant can be added to facilitate the formation of a homogeneoussolution or suspension.

We have found that the drug conjugates and compositions of the presentinvention are particularly effective in the treatment of cancer.

Thus, as described earlier, the present invention provides a method oftreating a patient in need thereof, notably a human, affected by cancerwhich comprises administering to the affected individual atherapeutically effective amount of a drug conjugate or a composition ofthe present invention. The present invention provides a drug conjugateaccording to the present invention for use in the treatment of cancer,and more preferably a cancer selected from lung cancer, colorectalcancer, breast cancer, pancreas carcinoma, kidney cancer, leukaemia,multiple myeloma, lymphoma, gastric and ovarian cancer. Most preferredcancer is breast cancer.

The drug conjugates and compositions of the present invention are usefulfor inhibiting the multiplication of a tumor cell or cancer cell, or fortreating cancer in an animal. The drug conjugates and compositions ofthe present invention can be used accordingly in a variety of settingsfor the treatment of animal cancers. The conjugates of the inventioncomprising Drug-Linker-Moiety comprising at least one antigen bindingsite can be used to deliver a Drug or Drug unit to a tumor cell orcancer cell. Without being bound by theory, in one embodiment, theMoiety comprising at least one antigen binding site of a drug conjugateof the present invention binds to or associates with a cancer-cell or atumor-cell-associated antigen, and the drug conjugate of the presentinvention can be taken up inside a tumor cell or cancer cell throughreceptor-mediated endocytosis. The antigen can be attached to a tumorcell or cancer cell or can be an extracellular matrix protein associatedwith the tumor cell or cancer cell. Once inside the cell, one or morespecific sequences within the Linker unit are hydrolytically cleaved byone or more tumor-cell or cancer-cell-associated proteases orhydrolases, resulting in release of a Drug or a Drug-Linker Compound.The released Drug or Drug-Linker Compound is then free to migrate in thecell and induce cytotoxic activities. In an alternative embodiment, theDrug or Drug unit is cleaved from the drug conjugate of the presentinvention outside the tumor cell or cancer cell, and the Drug orDrug-Linker Compound subsequently penetrates the cell.

In one embodiment, the Moiety comprising at least one antigen bindingsite binds to the tumor cell or cancer cell. In another embodiment, theMoiety comprising at least one antigen binding site binds to a tumorcell or cancer cell antigen which is on the surface of the tumor cell orcancer cell. In yet another embodiment, the Moiety comprising at leastone antigen binding site binds to a tumor cell or cancer cell antigenwhich is an extracellular matrix protein associated with the tumor cellor cancer cell.

The specificity of the Moiety comprising at least one antigen bindingsite for a particular tumor cell or cancer cell can be important fordetermining those tumors or cancers that are most effectively treated.For example, drug conjugates of the present invention having aTrastuzumab unit can be useful for treating antigen positive carcinomasincluding leukaemias, lung cancer, colon cancer, lymphomas (e.g.Hodgkin's disease, non-Hodgkin's Lymphoma), solid tumors such as,sarcoma and carcinomas, Multiple myeloma, kidney cancer and melanoma.The cancer may preferably be lung cancer, colorectal cancer, breastcancer, pancreas carcinoma, kidney cancer, leukaemia, multiple myeloma,lymphoma or ovarian cancer. For example, drug conjugates of the presentinvention having a Rituximab unit can be useful for treating CD-20expressing tumors such as haematological cancers including leukemias andlymphomas. For example, drug conjugates of the present invention havingan anti-CD4 antibody unit can be useful for treating CD-4 expressingtumors such as haematological cancers including lymphomas. For example,drug conjugates of the present invention having an anti-CD5 antibodyunit can be useful for treating CD-5 expressing tumors such ashaematological cancers including leukemias and lymphomas. For example,drug conjugates of the present invention having an anti-CD13 antibodyunit can be useful for treating CD-13 expressing tumors such ashaematological cancers including leukemias and lymphomas.

Other particular types of cancers that can be treated with drugconjugates of the present invention include, but are not limited to:blood-borne cancers including all forms of leukemia; lymphomas, such asHodgkin's disease, non-Hodgkin's Lymphoma and Multiple myeloma.

In particular, the drug conjugates and compositions of the presentinvention show excellent activity in the treatment of breast cancer.

Drug conjugates and compositions of the present invention provideconjugation specific tumor or cancer targeting, thus reducing generaltoxicity of these conjugates. The Linker units stabilize the drugantibody conjugates in blood, yet are cleavable by tumor-specificproteases and hydrolases within the cell, liberating a Drug.

The drug conjugates and compositions of the present invention can beadministered to an animal that has also undergone surgery as treatmentfor the cancer. In one embodiment of the present invention, theadditional method of treatment is radiation therapy.

In a specific embodiment of the present invention, the drug conjugate orcomposition of the present invention may be administered withradiotherapy. Radiotherapy may be administered at the same time, priorto or after treatment with the drug conjugate or composition of thepresent invention. In an embodiment, the drug conjugate or compositionof the present invention is administered concurrently with radiationtherapy. In another specific embodiment, the radiation therapy isadministered prior or subsequent to administration of a drug conjugateor composition of the present invention, preferably at least an hour,five hours, 12 hours, a day, a week, a month, more preferably severalmonths (e.g. up to three months), prior or subsequent to administrationof a drug antibody conjugate or composition of the present invention.

With respect to radiation, any radiation therapy protocol can be useddepending upon the type of cancer to be treated. For example, but not byway of limitation, x-ray radiation can be administered; in particular,high-energy megavoltage (radiation of greater that 1 MeV energy) can beused for deep tumors, and electron beam and orthovoltage x-ray radiationcan be used for skin cancers. Gamma-ray emitting radioisotopes, such asradioactive isotopes of radium, cobalt and other elements, can also beadministered.

In the present invention, there is provided a kit comprising atherapeutically effective amount of a drug conjugate according to thepresent invention and a pharmaceutically acceptable carrier. In anembodiment, there is provided a kit comprising a composition accordingto the present invention and, optionally, instructions for use in thetreatment of cancer, and more preferably a cancer selected from lungcancer, colorectal cancer, breast cancer, pancreas carcinoma, kidneycancer, leukaemia, multiple myeloma, lymphoma, gastric and ovariancancer.

In one embodiment, the kit according to this aspect is for use in thetreatment of cancer, and more preferably a cancer selected from lungcancer, colorectal cancer, breast cancer, pancreas carcinoma, kidneycancer, leukaemia, multiple myeloma, lymphoma, gastric and ovariancancer. Most preferred kit is for use in the treatment of breast cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is diagrammatically illustrated, by way of example, in theaccompanying drawings in which:

FIG. 1 is a schematic illustration of one process according to thepresent invention wherein conjugation to the antibody is via free thiolgroups;

FIG. 2 is a schematic illustration of one process according to thepresent invention wherein conjugation to the antibody is via free aminogroups.

FIG. 3 . Dose-response curves corresponding to one representativeexperiment conducted to evaluate the antiproliferative potential of theADC 1 in HER2-positive (black symbols) or HER2-negative (hollow symbols)cell lines. Dots correspond to the average of triplicates with errorbars denoting SD, drawing lines correspond to the best fitting bynonlinear regression of the experimental points to a four-parameterslogistic curve used to obtain the IC₅₀ values reported in Table 13.

FIG. 4 . Histograms showing the effect on cell survival of eitherTrastuzumab at 20 μg/mL or the ADC 1 at 16 or 2.5 μg/mL in several celllines, either HER2-positive (SK-BR-3 and HCC-1954) or HER2-negative(MDA-MB-231 and MCF-7). Bars correspond to the average of threedeterminations with error bars denoting SD. Statistical significance wasmeasured using an unpaired two-tailed t-test, p values are summarized asfollows:***, p<0.001; *, p<0.01; *, p<0.05.

FIG. 5 . Dose-response curves showing the antiproliferative potential ofthe ADC 2 in HER2-positive (black symbols) or HER2-negative (hollowsymbols) cell lines. Dots correspond to the average of triplicates witherror bars denoting SD, drawing lines correspond to the best fitting bynonlinear regression of the experimental points to a four-parameterslogistic curve used to obtain the IC₅₀ values reported in Table 15.

FIG. 6 . Histograms showing the effect on cell survival of eitherTrastuzumab at 20 μg/mL or the ADC 2 at 16 or 2.5 μg/mL in several celllines, either HER2-positive (SK-BR-3 and HCC-1954) or HER2-negative(MDA-MB-231 and MCF-7). Bars correspond to the average of threedeterminations with error bars denoting SD. Statistical significance wasmeasured using an unpaired two-tailed t-test, p values are summarized asfollows: ***, p<0.001; *, p<0.01; *, p<0.05.

FIG. 7 . Dose-response curves showing the antiproliferative potential ofthe ADC 3 in HER2-positive (black symbols) or HER2-negative (hollowsymbols) cell lines. Dots correspond to the average of triplicates witherror bars denoting SD, drawing lines correspond to the best fitting bynonlinear regression of the experimental points to a four-parameterslogistic curve used to obtain the IC₅₀ values reported in Table 17.

FIG. 8 . Histograms showing the effect on cell survival of eitherTrastuzumab at 20 μg/mL or the ADC 3 at 20 or 3 μg/mL in several celllines, either HER2-positive (SK-BR-3 and HCC-1954) or HER2-negative(MDA-MB-231 and MCF-7). Bars correspond to the average of threedeterminations with error bars denoting SD. Statistical significance wasmeasured using an unpaired two-tailed t-test, p values are summarized asfollows: ***, p<0.001; *, p<0.01; *, p<0.05.

FIG. 9 . Dose-response curves showing the antiproliferative potential ofthe ADC 4 in HER2-positive (black symbols) or HER2-negative (hollowsymbols) cell lines. Dots correspond to the average of triplicates witherror bars denoting SD, drawing lines correspond to the best fitting bynonlinear regression of the experimental points to a four-parameterslogistic curve used to obtain the IC50 values reported in Table 18.

FIG. 10 . Histograms showing the effect on cell survival of eitherTrastuzumab at 3 μg/mL or the ADC 4 at 3 or 0.2 μg/mL in several celllines, either HER2-positive (SK-BR-3 and HCC-1954) or HER2-negative(MDA-MB-231 and MCF-7). Bars correspond to the average of threedeterminations with error bars denoting SD. Statistical significance wasmeasured using an unpaired two-tailed t-test, p values are summarized asfollows: ***, p<0.001; *, p<0.01; *, p<0.05.

FIG. 11 . Dose-response curves showing the antiproliferative potentialof the ADC 6 in HER2-positive (black symbols) or HER2 negative (hollowsymbols) cell lines. Dots correspond to the average of triplicates witherror bars denoting SD, drawing lines correspond to the best fitting bynonlinear regression of the experimental points to a four-parameterslogistic curve used to obtain the IC₅₀ values reported in Table 21.

FIG. 12 . Tumor volume evaluation of BT-474 tumors in mice treated withplacebo, 11-R (at 5 mg/kg), ADC 1 (2.2 TCEP) (at 1.6 and 6.5 mg/kg) andADC 1 (at 2.24 mg/kg).

FIG. 13 . Tumor volume evaluation of JIMT-1 tumors in mice treated withplacebo, 11-R (at 5.0 mg/kg) and ADC 1 (at 5, 10 and 30 mg/kg).

FIG. 14 . Tumor volume evaluation of SKOV3 tumors in mice treated withplacebo, 11-R (at 5.0 mg/kg) and ADC 1 (at 5, 10 and 30 mg/kg).

FIG. 15 . Tumor volume evaluation of N87 tumors in mice treated withplacebo, 11-R (at 5 mg/kg) and ADC 1 (2.2 TCEP) (at 5, 10 and 30 mg/kg)—

FIG. 16 Tumor volume evaluation of Gastric-008 (PDX) tumors in micetreated with placebo, 11-R (at 5 mg/kg) and ADC-1 (at 5, 10 and 30mg/kg).

EXAMPLES

The present invention is further illustrated by way of the following,non-limiting examples. In the examples, the following abbreviations areused:

-   CDI, 1,1′-Carbonyldiimidazole-   DIPEA, N,N-Diisopropylethylamine-   Hex, Hexane-   EtOAc, Ethyl acetate-   DCM, Dichloromethane-   NMP, N-Methyl-2-pyrrolidone-   DMF, Dimethylformamide-   EDC, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride-   EDTA, Ethylenediaminetetraacetic acid-   MeOH, Methanol-   DTT, Dithiothreitol-   Py, Pyridine-   THF, Tetrahydrofuran-   TCEP, Tris[2-carboxyethyl]phosphine hydrochloride-   MC, 6-Maleimidocaproyl-   Fmoc, 9-Fluorenylmethoxycarbonyl-   Cit, Citrulline-   Val, Valine-   DMSO, Dimethylsulfoxide-   Trt, Triphenylmethyl-   HOBt, 1-Hydroxybenzotriazole-   DIPCDl, N,N′-Diisopropylcarbodiimide-   TFA, Trifluoroacetic acid-   PABOH, 4-Aminobenzyl alcohol-   bis-PNP, bis(4-Nitrophenyl) carbonate-   NAC, N-Acetylcysteine-   SEC, Size-Exclusion Chromatography-   HPLC, High Performance Liquid Chromatography-   ADC, Antibody Drug Conjugate-   ATCC, American Type Culture Collection-   DMEM, Dulbecco's Modified Eagle's Medium-   RPMI, Rosmell Park Memorial Institute Medium-   ITS, Insulin-transferrin-sodium selenite media supplement-   FCS, Fetal Calf Serum-   SRB, Sulforhodamine B-   PBS, Phosphate Buffered Saline-   DR, Dose-Response-   UV, Ultraviolet-   SMCC, Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate-   LAR, Linker to Antibody Ratio

Synthesis of Compounds

Compound 1 was prepared as described in Example 20 of WO 01/87895.

Example 0-1 A)

To a solution of 1 (0.5 g, 0.80 mmol) in acetic acid (20 mL, 0.04 M) wasadded L-tryptophanol (2-S) (533 mg, 3.0 mmol, Sigma-Aldrich). Thereaction mixture was stirred at 23° C. for 16 h and then acetic acid wasevaporated. An aqueous saturated solution of NaHCO₃ was added and themixture was extracted with CH₂Cl₂. The combined organic layers weredried over anhydrous Na₂SO₄, filtered and concentrated under vacuum.Flash chromatography (Hexane:EtOAc, 1:1) gave compounds 3-S (616 mg,97%) and 3a-S (12 mg, 2%).

3-S

R_(f)=0.50 (Hexane:EtOAc, 1:1).

¹H NMR (300 MHz, CDCl₃): δ 7.71 (s, 1H), 7.36 (dd, J=7.9, 1.0 Hz, 1H),7.27 (dd, J=8.2, 0.9 Hz, 1H), 7.13 (ddd, J=8.3, 7.0, 1.2 Hz, 1H), 7.03(ddd, J=8.0, 7.0, 1.0 Hz, 1H), 6.62 (s, 1H), 6.26 (d, J=1.4 Hz, 1H),6.04 (d, J=1.3 Hz, 1H), 5.75 (s, 1H), 5.14 (dd, J=11.7, 1.2 Hz, 1H),4.60 (s, 1H), 4.41 (s, 1H), 4.36-4.24 (m, 2H), 4.21 (d, J=2.7 Hz, 1H),3.82 (s, 3H), 3.52 (s, 1H), 3.50-3.47 (m, 1H), 3.45 (dq, J=8.4, 2.2 Hz,1H), 3.35 (t, J=10.1 Hz, 1H), 3.01-2.78 (m, 5H), 2.62 (dd, J=15.3, 4.7Hz, 1H), 2.41 (s, 1H), 2.38 (s, 3H), 2.37-2.31 (m, 1H), 2.28 (s, 3H),2.17 (s, 3H), 2.06 (s, 3H).

ESI-MS m/z: 794.2 (M+H)⁺.

3a-S

R_(f)=0.70 (Hexane:EtOAc, 1:1).

¹H NMR (500 MHz, CDCl₃): δ 7.83 (s, 1H), 7.38 (dt, J=7.9, 0.9 Hz, 1H),7.25 (dt, J=8.3, 0.9 Hz, 1H), 7.11 (ddd, J=8.2, 7.1, 1.2 Hz, 1H), 7.02(ddd, J=8.0, 7.0, 1.0 Hz, 1H), 6.62 (s, 1H), 6.24 (d, J=1.4 Hz, 1H),6.03 (d, J=1.3 Hz, 1H), 5.79 (s, 1H), 5.13 (d, J=11.7 Hz, 1H), 4.60 (s,1H), 4.39 (s, 1H), 4.36-4.22 (m, 3H), 4.17-4.09 (m, 1H), 3.91 (dd,J=10.5, 8.6 Hz, 1H), 3.83 (s, 3H), 3.51-3.41 (m, 2H), 3.04-2.92 (m, 3H),2.72 (dd, J=15.1, 4.0 Hz, 1H), 2.54-2.41 (m, 2H), 2.38 (s, 3H),2.35-2.30 (m, 1H), 2.29 (s, 3H), 2.21-2.16 (m, 1H), 2.18 (s, 3H), 2.12(s, 3H); 2.05 (s, 3H).

¹³C NMR (101 MHz, CDCl₃): δ 171.2, 170.7, 168.6, 147.5, 145.8, 143.0,141.1, 140.4, 135.6, 130.1, 129.5, 126.7, 122.2, 121.2, 120.9, 119.4,118.4, 118.2, 118.2, 113.6, 113.5, 110.9, 110.0, 109.1, 102.1, 91.4,67.2, 63.4, 61.3, 60.4, 59.7, 59.1, 54.8, 54.6, 47.7, 42.0, 41.6, 31.6,24.0, 22.6, 21.0, 15.9, 14.2, 9.7.

ESI-MS m/z: 836.2 (M+H)⁺.

B)

To a solution of 3-S (616 mg, 0.77 mmol) in CH₃CN:H₂O (1.39:1, 51 mL,0.015 M) was added AgNO₃ (3.40 g, 23.3 mmol). After 3 h at 23° C., thereaction mixture was quenched with a mixture 1:1 of saturated aqueoussolutions of NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂,stirred for 5 min, and extracted with CH₂Cl₂. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to give 4-S (471 mg, 78%).

R_(f)=0.50 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (500 MHz, CDCl₃): δ 7.71 (s, 1H), 7.36 (dd, J=7.8, 1.1 Hz, 1H),7.26 (dd, J=7.8, 1.1 Hz, 1H), 7.12 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 7.03(ddd, J=8.0, 7.1, 1.0 Hz, 1H), 6.64 (s, 1H), 6.23 (d, J=1.3 Hz, 1H),6.01 (d, J=1.4 Hz, 1H), 5.75 (s, 1H), 5.25 (d, J=11.4 Hz, 1H), 4.92 (s,1H), 4.52 (br s, 3H), 4.22 (dd, J=11.4, 2.2 Hz, 1H), 4.19 (s, 1H), 3.83(s, 3H), 3.54 (br s, 2H), 3.35 (t, J=10.2 Hz, 1H), 3.26 (s, 1H),3.01-2.93 (m, 3H), 2.88 (br s, 3H), 2.63 (dd, J=15.2, 4.8 Hz, 1H), 2.38(s, 3H), 2.36-2.31 (m, 2H), 2.28 (s, 3H), 2.05 (s, 3H).

¹³C NMR (126 MHz, CDCl₃): δ 171.9, 168.6, 147.5, 145.4, 142.9, 141.2,140.7, 135.5, 130.4, 126.8, 122.3, 122.0, 121.3, 119.4, 118.4, 115.2,112.8, 111.0, 110.0, 109.6, 101.8, 81.9, 76.8, 65.2, 62.8, 62.5, 60.4,58.1, 57.9, 55.9, 55.1, 53.4, 51.6, 41.8, 41.3, 39.6, 24.1, 23.8, 20.5,15.8, 9.7.

ESI-MS m/z: 767.3 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z 767.2788 [M−H₂O+H]⁺ (Calcd. for C41H₄₃N₄O₉S:767.2745).

B′)

To a solution of 3a-S (30 mg, 0.035 mmol) in CH₃CN:H₂O (1.39:1, 2.4 mL,0.015 M) was added AgNO₃ (180 mg, 1.07 mmol). After 3 h at 23° C., thereaction mixture was quenched with a mixture 1:1 of saturated aqueoussolutions of NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂,stirred for 5 min, and extracted with CH₂Cl₂. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to give 4a-S (24 mg, 83%).

R_(f)=0.60 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.81 (s, 1H), 7.37 (d, J=7.8 Hz, 1H),7.30-7.21 (m, 1H), 7.06 (dddt, J=34.7, 8.0, 7.1, 1.1 Hz, 2H), 6.63 (s,1H), 6.22 (d, J=1.3 Hz, 1H), 6.02 (dd, J=12.9, 1.4 Hz, 1H), 5.74 (s,1H), 5.25-5.21 (m, 1H), 4.89 (d, J=8.7 Hz, 1H), 4.55-4.45 (m, 2H),4.30-4.18 (m, 1H), 4.14 (dd, J=10.5, 4.2 Hz, 1H), 4.00-3.88 (m, 2H),3.82 (s, 3H), 3.56-3.44 (m, 2H), 3.23 (d, J=9.0 Hz, 1H), 2.95 (d, J=15.7Hz, 2H), 2.87-2.78 (m, 2H), 2.71 (dd, J=15.0, 3.9 Hz, 1H), 2.48 (dd,J=15.1, 9.6 Hz, 1H), 2.37 (s, 3H), 2.35-2.29 (m, 1H), 2.28 (s, 3H),2.22-2.16 (m, 1H), 2.15 (s, 3H), 2.12 (s, 3H), 2.03 (s, 3H).

ESI-MS m/z: 809.2 (M−H₂O+H)⁺.

Example 0-2 A)

To a solution of 1 (0.5 g, 0.80 mmol) in acetic acid (20 mL, 0.04 M) wasadded D-tryptophanol (2-R) (533 mg, 3.0 mmol, Sigma-Aldrich). Thereaction mixture was stirred at 23° C. for 16 h and then acetic acid wasevaporated. An aqueous saturated solution of NaHCO₃ was added and themixture was extracted with CH₂Cl₂. The combined organic layers weredried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum.Flash chromatography (Hexane:EtOAc, 1:1) gave compound 3-R (479 mg,75%).

R_(f)=0.44 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.61 (s, 1H), 7.39 (d, J=7.8 Hz, 1H), 7.29(d, J=9.6 Hz, 1H), 7.12 (t, J=7.3 Hz, 1H), 7.03 (t, J=7.3 Hz, 1H), 6.60(s, 1H), 6.25 (s, 1H), 6.03 (s, 1H), 5.75 (s, 1H), 5.04 (d, J=11.7 Hz,1H), 4.62 (s, 1H), 4.37 (s, 1H), 4.32-4.25 (m, 1H), 4.22 (d, J=2.7 Hz,1H), 4.19-4.09 (m, 1H), 3.82 (s, 3H), 3.77 (s, 1H), 3.64 (d, J=9.0 Hz,1H), 3.49-3.41 (m, 2H), 3.02-2.90 (m, 2H), 2.60-2.52 (m, 2H), 2.45 (d,J=14.7 Hz, 2H), 2.40 (s, 3H), 2.28 (s, 3H), 2.22-2.14 (m, 2H), 2.18 (s,3H), 2.10 (m, 3H).

ESI-MS m/z: 794.3 (M+H)⁺.

B)

To a solution of 3-R (479 mg, 0.60 mmol) in CH₃CN:H₂O (1.39:1, 40 mL,0.015 M) was added AgNO₃ (3.03 g, 18.1 mmol). After 3 h at 23° C., thereaction mixture was quenched with a mixture 1:1 of saturated aqueoussolutions of NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂,stirred for 5 min, and extracted with CH₂Cl₂. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to afford 4-R (428 mg, 91%).

R_(f)=0.45 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.62 (s, 1H), 7.39 (d, J=8.1 Hz, 1H), 7.28(d, J=8.1 Hz, 1H), 7.11 (ddd, J=8.2, 7.0, 1.2 Hz, 1H), 7.02 (ddd, J=7.9,7.1, 1.0 Hz, 1H), 6.61 (s, 1H), 6.22 (d, J=1.3 Hz, 1H), 5.99 (d, J=1.3Hz, 1H), 5.73 (s, 1H), 5.17 (dd, J=11.5, 1.2 Hz, 1H), 4.86 (s, 1H),4.56-4.47 (m, 2H), 4.17 (dd, J=5.1, 1.6 Hz, 1H), 4.08 (dd, J=11.5, 2.1Hz, 1H), 3.81 (s, 3H), 3.78 (d, J=3.8 Hz, 1H), 3.64 (dd, J=10.8, 3.8 Hz,2H), 3.51 (d, J=5.1 Hz, 1H), 3.48-3.43 (m, 2H), 3.24 (d, J=8.6 Hz, 1H),3.00-2.80 (m, 2H), 2.57 (s, 1H), 2.55-2.43 (m, 1H), 2.40 (s, 3H), 2.27(s, 3H), 2.19-2.12 (m, 1H), 2.16 (s, 3H), 2.08 (s, 3H).

¹³C NMR (101 MHz, CDCl₃): δ 171.8, 168.6, 147.6, 145.4, 143.0, 141.3,140.7, 136.0, 131.1, 130.0, 129.6, 126.6, 122.1, 121.6, 121.2, 119.4,118.4, 115.6, 112.9, 111.1, 110.6, 101.8, 81.7, 65.8, 62.7, 61.8, 60.4,60.3, 57.9, 57.8, 56.1, 55.0, 52.1, 42.2, 41.3, 41.1, 23.8, 23.4, 20.5,15.7, 9.8.

ESI-MS m/z: 767.6 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 767.2799 [M−H₂O+H]⁺ (Calcd. for C41H₄₃N₄O₉S:767.2745).

Example 0-3. Synthesis of allylN—[(R)-(2-amino-3-(1H-indol-3-yl)propyl)]carbamate (9-R)

A)

To a solution of D-tryptophanol (2-R) (2.0 g, 10.4 mmol) in CH₃CN (42mL, 4 mL/mmol) was added di-tert-butyl dicarbonate (4.6 g, 20.8 mmol).The reaction mixture was stirred at 23° C. for 3 h and concentratedunder vacuum. Flash chromatography (CH₂Cl₂:CH₃OH from 99:1 to 85:15) toafford 5-R (2.2 g, 73%).

R_(f)=0.5 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 8.13 (s, 1H), 7.67 (dd, J=7.8, 1.1 Hz, 1H),7.38 (dd, J=8.1, 1.3 Hz, 1H), 7.29-7.10 (m, 2H), 7.06 (s, 1H), 4.82 (s,1H), 4.00 (s, 1H), 3.71 (dd, J=11.0, 3.8 Hz, 1H), 3.62 (dd, J=11.0, 5.5Hz, 1H), 3.01 (d, J=6.7 Hz, 2H), 2.14 (s, 1H), 1.44 (s, 9H).

B)

To a solution of 5-R (2.4 g, 8.2 mmol) in CH₂Cl₂ (50 mL, 6 mL/mmol) wasadded phthalimide (2.7 g, 18.2 mmol), triphenylphosphine (4.8 g, 18.2mmol) and the mixture was cooled at 0° C. A solution of diethylazodicarboxylate (DEAD) solution in CH₂Cl₂ (25 mL, 3 mL/mmol) was addedfor 15 min. The reaction was stirred at 23° C. for 16 h, concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to afford 6-R (3.3 g, 96%).

R_(f)=0.7 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 8.50 (s, 1H), 7.81 (dd, J=5.5, 3.1 Hz, 2H),7.66 (dd, J=5.6, 3.2 Hz, 2H), 7.60 (d, J=7.8 Hz, 1H), 7.35 (d, J=8.0 Hz,1H), 7.19-7.04 (m, 3H), 4.81 (s, 1H), 4.40 (s, 1H), 3.83 (dd, J=13.9,3.7 Hz, 1H), 3.72 (dd, J=13.9, 9.9 Hz, 1H), 3.08-3.01 (m, 2H), 1.23 (s,9H).

C)

To a solution of 6-R (3.25 g, 7.74 mmol) in ethanol (231 mL, 30 mL/mmol)was added hydrazine monohydrate (37 mL, 774 mmol). The reaction mixturewas stirred at 80° C. in sealed tube for 2.25 h, concentrated undervacuum. Flash chromatography (EtOAc:CH₃OH, from 100:1 to 50:50) afforded7-R (2.15 g, 96%).

R_(f)=0.2 (EtOAc:CH₃OH, 6:4).

¹H NMR (400 MHz, CD3OD): δ 7.60 (d, J=7.9 Hz, 1H), 7.33 (d, J=8.1 Hz,1H), 7.13-7.04 (m, 2H), 7.05-6.96 (m, 1H), 4.02-3.94 (m, 1H), 2.99-2.87(m, 3H), 2.78 (dd, J=13.1, 9.7 Hz, 1H), 1.39 (s, 9H).

ESI-MS m/z: 290.2 (M+H)⁺.

D)

To a solution of 7-R (2.15 g, 7.4 mmol) in CH₃CN (74 mL, 10 mL/mmol) andDMF (7.4 mL, 1 mL/mmol) was added N,N-diisopropylethylamine (1.06 mL,5.9 mmol) and allyl chloroformate (7.9 mL, 74 mmol). The reaction wasstirred at 23° C. for 16 h. The mixture was diluted with EtOAc, NH₄Clwas added and the mixture was extracted with EtOAc. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(Hexane:EtOAc, from 100:1 to 1:100) to afford 8-R (1.69 g, 61%).

R_(f)=0.4 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 8.25 (s, 1H), 7.62 (d, J=7.8 Hz, 1H), 7.35(dd, J=8.1, 0.9 Hz, 1H), 7.16 (dddd, J=27.8, 8.0, 7.0, 1.1 Hz, 2H), 7.04(d, J=2.4 Hz, 1H), 5.90 (ddt, J=17.3, 10.7, 5.6 Hz, 1H), 5.34-5.22 (m,1H), 5.20 (dt, J=10.5, 1.4 Hz, 1H), 5.12 (s, 1H), 4.82 (s, 1H), 4.55(dq, J=5.4, 1.7 Hz, 2H), 4.02 (s, 1H), 3.35 (dt, J=10.0, 4.7 Hz, 1H),3.21 (s, 1H), 2.95 (ddd, J=21.6, 15.4, 9.1 Hz, 2H), 1.42 (s, 9H).

ESI-MS m/z: 274.3 (M−Boc+H)⁺.

To a solution of 8-R (1.30 g, 3.50 mmol) in CH₂Cl₂ (58 mL, 16.6 mL/mmol)was added trifluoroacetic acid (30 mL, 8.3 mL/mmol). The reactionmixture was stirred at 23° C. for 1.5 h, concentrated under vacuum togive crude 9-R which was used in the next steps without furtherpurification.

R_(f)=0.2 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.95 (s, 1H), 7.53 (d, J=7.8 Hz, 1H), 7.36(d, J=8.1 Hz, 1H), 7.17 (s, 1H), 7.09 (t, J=7.5 Hz, 1H), 7.03 (t, J=7.5Hz, 1H), 5.87 (ddt, J=16.4, 10.8, 5.6 Hz, 1H), 5.34-5.13 (m, 2H), 4.50(d, J=5.5 Hz, 2H), 3.62 (bs, 1H), 3.42 (dd, J=14.9, 3.9 Hz, 1H),3.36-3.20 (m, 1H), 3.11-3.00 (m, 2H).

ESI-MS m/z: 274.3 (M+H)⁺.

Example 0-4. Synthesis of allylN—[(S)-(2-amino-3-(1H-indol-3-yl)propyl)]carbamate (9-S)

A)

To a solution of L-tryptophanol (2-S) (2.0 g, 10.4 mmol) in CH₃CN (42mL, 4 mL/mmol) was added Di-tert-butyl dicarbonate (4.6 g, 20.8 mmol).The reaction mixture was stirred at 23° C. for 3 h, concentrated undervacuum. Flash chromatography (CH₂Cl₂:CH₃OH, from 99:1 to 85:15) toafford 5-S (2.24 g, 73%).

R_(f)=0.5 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 8.10 (s, 1H), 7.65 (dd, J=7.8, 1.1 Hz, 1H),7.37 (dd, J=8.1, 1.3 Hz, 1H), 7.23-7.11 (m, 2H), 7.06 (s, 1H), 4.81 (s,1H), 3.99 (s, 1H), 3.70 (dd, J=11.0, 3.8 Hz, 1H), 3.61 (dd, J=11.0, 5.5Hz, 1H), 3.00 (d, J=6.7 Hz, 2H), 2.01 (s, 1H), 1.42 (s, 9H).

B)

To a solution of 5-S (1.2 g, 4.13 mmol) in CH₂Cl₂ (24.8 mL, 6 mL/mmol)was added phthalimide (1.33 g, 9.1 mmol), triphenylphosphine (2.4 g, 9.1mmol) and the mixture was cooled at 0° C. A solution of diethylazodicarboxylate (DEAD) solution (3 mL, 10.32 mmol) in CH₂Cl₂ (12.4 mL,3 mL/mmol) was added for 15 min. The reaction was stirred at 23° C. for16 h, concentrated under vacuum. The residue obtained was purified byflash chromatography (CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to afford 6-S(2.8 g, >100%).

R_(f)=0.7 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 8.49 (s, 1H), 7.80 (dd, J=5.4, 3.1 Hz, 2H),7.66 (dd, J=5.6, 3.2 Hz, 2H), 7.60 (d, J=7.8 Hz, 1H), 7.34 (d, J=8.0 Hz,1H), 7.21-7.04 (m, 3H), 4.74 (s, 1H), 4.42 (s, 1H), 3.83 (dd, J=13.9,3.7 Hz, 1H), 3.72 (dd, J=13.9, 9.9 Hz, 1H), 3.10-3.01 (m, 2H), 1.23 (s,9H).

C)

To a solution of 6-S (0.86 g, 2.07 mmol) in ethanol (72 mL, 36 mL/mmol)was added hydrazine monohydrate (10 mL, 207 mmol). The reaction mixturewas stirred at 80° C. in sealed tube for 2.25 h, concentrated undervacuum. Flash chromatography (EtOAc:CH₃OH, from 100:1 to 50:50) toafford 7-S (1.0 g, 84%).

R_(f)=0.2 (EtOAc:CH₃OH, 6:4).

¹H NMR (400 MHz, CD3OD): δ 7.61 (d, J=7.9 Hz, 1H), 7.35 (d, J=8.1 Hz,1H), 7.13-6.97 (m, 2H), 7.09 (s, 1H), 4.06-3.96 (m, 1H), 3.01-2.76 (m,4H), 1.38 (s, 9H).

ESI-MS m/z: 290.3 (M+H)⁺.

D)

To a solution of 7-S (0.95 g, 3.3 mmol) in CH₃CN (33 mL, 10 mL/mmol) andDMF (3.3 mL, 1 mL/mmol) was added N,N-diisopropylethylamine (0.5 mL, 2.6mmol) and allyl chloroformate (3.5 mL, 33 mmol). The reaction wasstirred at 23° C. for 20 h. The mixture was diluted with EtOAc, NH₄Clwas added and the mixture was extracted with EtOAc. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(Hexane:EtOAc, from 100:1 to 1:100) to afford 8-S (0.88 g, 73%).

R_(f)=0.5 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 8.17 (s, 1H), 7.63 (d, J=7.8 Hz, 1H), 7.20(dd, J=8.1, 0.9 Hz, 1H), 7.13 (dddd, J=27.8, 8.0, 7.0, 1.1 Hz, 2H), 7.06(d, J=2.4 Hz, 1H), 5.90 (ddt, J=17.3, 10.7, 5.6 Hz, 1H), 5.31-5.18 (m,2H), 5.09 (s, 1H), 4.80 (s, 1H), 4.59-4.52 (m, 2H), 4.03 (s, 1H), 3.37(dt, J=10.0, 4.7 Hz, 1H), 3.21 (s, 1H), 3.05-2.87 (m, 2H), 1.42 (s, 9H).

ESI-MS m/z: 274.3 (M−Boc+H)⁺.

E)

To a solution of 8-S (0.875 g, 2.3 mmol) in CH₂Cl₂ (38 mL, 16.6 mL/mmol)was added trifluoroacetic acid (19 mL, 8.3 mL/mmol). The reactionmixture was stirred at 23° C. for 2 h, concentrated under vacuum to givecrude 9-S which was used in the next steps without further purification.

R_(f)=0.2 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CD3OD): δ 7.56 (d, J=7.8 Hz, 1H), 7.37 (d, J=8.1 Hz,1H), 7.21 (s, 1H), 7.13 (t, J=7.5 Hz, 1H), 7.05 (t, J=7.5 Hz, 1H), 5.94(ddt, J=16.4, 10.8, 5.6 Hz, 1H), 5.34-5.16 (m, 2H), 4.56 (d, J=5.5 Hz,2H), 3.60 (bs, 1H), 3.43 (dd, J=14.9, 3.9 Hz, 1H), 3.37-3.31 (m, 1H),3.14-2.99 (m, 2H).

ESI-MS m/z: 274.3 (M+H)⁺.

Example 0-5 A)

To a solution of 1 (1.45 g, 2.33 mmol) in acetic acid (58 mL, 0.08 M)was added 9-R (0.95 g, 3.50 mmol). The reaction mixture was stirred a50° C. for 18 h and then acetic acid was evaporated. An aqueoussaturated solution of NaHCO₃ was added and the mixture was extractedwith CH₂Cl₂. The combined organic layers were dried over anhydrousNa₂SO₄. Flash chromatography (Hexane:EtOAc, 1:1) gives compound 10-R(1.3 g, 64%).

R_(f)=0.5 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.66 (s, 1H), 7.36 (d, J=7.9 Hz, 1H), 7.27(d, J=7.9 Hz, 1H), 7.10 (ddd, J=8.3, 7.0, 1.3 Hz, 1H), 7.01 (td, J=7.5,7.0, 1.0 Hz, 1H), 6.62 (s, 1H), 6.23 (d, J=1.4 Hz, 1H), 6.01 (d, J=1.4Hz, 1H), 5.99-5.89 (m, 1H), 5.79 (s, 1H), 5.44-5.21 (m, 2H), 5.14-4.99(m, 2H), 4.63 (ddd, J=7.3, 4.4, 1.5 Hz, 2H), 4.36 (s, 1H), 4.33-4.24 (m,1H), 4.29-4.26 (m, 1H), 4.21 (d, J=2.7 Hz, 1H), 4.19-4.13 (m, 3H), 3.80(s, 3H), 3.56 (s, 1H), 3.48-3.43 (m, 3H), 3.27 (dt, J=13.2, 4.0 Hz, 1H),3.04-2.88 (m, 2H), 2.56 (dd, J=15.2, 3.8 Hz, 1H), 2.49-2.35 (m, 2H),2.31 (s, 3H), 2.28 (s, 3H), 2.17 (s, 3H), 2.07 (s, 3H).

ESI-MS m/z: 877.3 (M+H)⁺.

B)

To a solution of 10-R (600 mg, 0.68 mmol) in CH₂Cl₂ (12 mL, 18 mL/mmol)was added bis(triphenylphosphine)palladium(II) dichloride (77 mg, 0.1mmol) and acetic acid (0.4 mL, 6.8 mmol). Tributyltin hydride (1.1 mL,4.08 mmol) was added at 0° C., the reaction mixture was stirred at 0° C.for 0.5 h and concentrated under vacuum. The crude obtained was dilutedwith EtOAc, saturated aqueous solution of NH₄Cl was added, and themixture was extracted with EtOAc. The combined organic layers were driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Flashchromatography (Hexane:EtOAc, from 100:1 to 1:100 and EtOAc:CH₃OH, from100:1 to 1:100) to afford 11-R (440 mg, 82%).

R_(f)=0.5 (CH₂Cl₂:CH₃OH, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.64 (s, 1H), 7.38 (d, J=7.9 Hz, 1H), 7.29(d, J=8.1 Hz, 1H), 7.11 (ddt, J=8.3, 7.0, 1.4 Hz, 1H), 7.03 (ddt, J=8.3,7.0, 1.4 Hz, 1H), 6.58 (s, 1H), 6.24 (d, J=1.5 Hz, 1H), 6.02 (d, J=1.5Hz, 1H), 5.02 (d, J=11.8 Hz, 1H), 4.63 (s, 1H), 4.36 (s, 1H), 4.28 (d,J=5.1 Hz, 1H), 4.21 (d, J=2.2 Hz, 1H), 4.16 (s, 1H), 3.80 (s, 3H),3.51-3.39 (m, 4H), 3.32-3.13 (m, 3H), 2.95 (d, J=8.9 Hz, 2H), 2.89-2.76(m, 2H), 2.73-2.57 (m, 1H), 2.42 (d, J=14.8 Hz, 1H), 2.36 (s, 3H), 2.25(s, 3H), 2.16 (s, 3H), 2.09 (s, 3H).

ESI-MS m/z: 793.2 (M+H)⁺.

C)

To a solution of 11-R (850 mg, 1.07 mmol) in CH₃CN:H₂O (1.39:1, 70 mL,0.015 M) was added AgNO₃ (3.64 g, 21.4 mmol). After 17 h at 23° C., thereaction was quenched with a mixture 1:1 of saturated aqueous solutionsof NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂, stirred for5 min, and extracted with CH₂Cl₂. The combined organic layers were driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Theresidue obtained was purified by flash chromatography (CH₂Cl₂:CH₃OH,from 99:1 to 85:15) to give 12-R (553 mg, 66%).

R_(f)=0.3 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (500 MHz, CDCl₃): δ 7.60 (s, 1H), 7.38 (d, J=7.9 Hz, 1H), 7.28(d, J=7.9 Hz, 1H), 7.11 (ddt, J=8.3, 7.1, 1.2 Hz, 1H), 7.02 (ddt, J=8.3,7.1, 1.2 Hz, 1H), 6.58 (s, 1H), 6.22 (s, 1H), 6.00 (s, 1H), 5.16 (d,J=11.5 Hz, 1H), 4.87 (s, 1H), 4.54 (s, 1H), 4.51 (d, J=3.3 Hz, 1H), 4.17(d, J=5.4 Hz, 1H), 4.07 (dd, J=11.3, 2.2 Hz, 1H), 3.81 (s, 3H), 3.52 (d,J=5.1 Hz, 1H), 3.24 (d, J=8.8 Hz, 2H), 2.99-2.78 (m, 4H), 2.66 (dd,J=14.9, 3.5 Hz, 1H), 2.49-2.39 (m, 2H), 2.38 (s, 3H), 2.28 (m, 2H), 2.25(s, 3H), 2.21-2.16 (m, 2H), 2.15 (s, 3H), 2.08 (s, 3H).

¹³C NMR (101 MHz, CD3OD): δ 171.7, 169.4, 148.7, 145.9, 143.7, 141.4,140.9, 136.9, 130.8, 130.0, 129.7, 126.0, 121.4, 121.0, 119.7, 119.1,118.4, 117.5, 114.9, 110.8, 107.5, 106.4, 102.1, 91.3, 63.2, 60.0, 59.0,58.6, 55.3, 54.6, 52.7, 52.4, 48.4, 45.8, 42.5, 40.2, 24.5, 23.2, 19.2,15.0, 8.2.

ESI-MS m/z: 766.2 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 766.2972 [M−H₂O+H]⁺ (Calcd. for C₄₁H₄₄N₅O₈S+:766.2905).

C′)

To a solution of 10-R (700 mg, 0.8 mmol) in CH₃CN:H₂O (1.39:1, 52.5 mL,0.015 M) was added AgNO₃ (2.66 g, 16 mmol). After 20 h at 23° C., thereaction mixture was quenched with a mixture 1:1 of saturated aqueoussolutions of NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂,stirred for 5 min, and extracted with CH₂Cl₂. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to give 13-R (438 mg, 63%).

R_(f)=0.40 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.64 (s, 1H), 7.37 (d, J=7.9 Hz, 1H),7.32-7.20 (m, 1H), 7.11 (t, J=7.7 Hz, 1H), 7.01 (t, J=7.4 Hz, 1H), 6.62(s, 1H), 6.21 (s, 1H), 6.05-5.90 (m, 1H), 5.99 (s, 1H), 5.75 (d, J=6.0Hz, 1H), 5.40-5.07 (m, 4H), 4.88 (d, J=14.7 Hz, 1H), 4.68-4.50 (m, 3H),4.28-4.13 (m, 1H), 4.08 (dt, J=11.4, 2.4 Hz, 1H), 3.83 (s, 3H),3.68-3.40 (m, 4H), 3.37-3.19 (m, 2H), 2.98-2.79 (m, 2H), 2.59-2.36 (m,3H), 2.29 (s, 3H), 2.27 (s, 3H), 2.14 (s, 3H), 2.10-2.16 (m, 1H), 2.08(s, 3H).

ESI-MS m/z: 850.3 (M−H₂O+H)⁺.

Example 0-6 A)

To a solution of 1 (955 mg, 1.5 mmol) in acetic acid (37.5 mL, 0.08 M)was added 9-S (627 mg, 2.29 mmol). The reaction mixture was stirred a50° C. for 18 h and then acetic acid was evaporated. An aqueoussaturated solution of NaHCO₃ was added and the mixture was extractedwith CH₂Cl₂. The combined organic layers were dried over anhydrousNa₂SO₄. Flash chromatography (Hexane:EtOAc, 1:1) gives compound 10-S(756 mg, 58%).

R_(f)=0.4 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.78 (s, 1H), 7.36 (d, J=7.9 Hz, 1H), 7.24(d, J=7.9 Hz, 1H), 7.10 (ddd, J=8.3, 7.0, 1.3 Hz, 1H), 7.01 (td, J=7.5,7.0, 1.0 Hz, 1H), 6.68 (s, 1H), 6.23 (d, J=1.4 Hz, 1H), 6.01 (d, J=1.4Hz, 1H), 6.07-5.93 (m, 1H), 5.82 (s, 1H), 5.41-5.19 (m, 2H), 5.1 (d,J=11.7 Hz, 1H), 4.66 (dt, J=5.9, 1.3 Hz, 1H), 4.57 (s, 1H), 4.37 (s,1H), 4.33-4.20 (m, 3H), 3.81 (s, 3H), 3.46 (d, J=4.2 Hz, 2H), 3.22-3.13(m, 1H), 3.11-2.88 (m, 4H), 2.66 (dd, J=15.2, 4.2 Hz, 1H), 2.51 (dd,J=15.3, 6.0 Hz, 1H), 2.43-2.32 (m, 2H), 2.31 (s, 3H), 2.26 (s, 3H), 2.19(s, 3H), 2.04 (s, 3H).

ESI-MS m/z: 877.3 (M+H)⁺.

B)

To a solution of 10-S (650 mg, 0.72 mmol) in CH₂Cl₂ (13.3 mL, 18mL/mmol) was added bis(triphenylphosphine)palladium(II) dichloride (83mg, 0.11 mmol) and acetic acid (0.42 mL, 7.4 mmol). Tributyltin hydride(1.2 mL, 4.4 mmol) was added at 0° C., the reaction mixture was stirredat 23° C. for 0.5 h, and concentrated under vacuum. Flash chromatography(Hexane:EtOAc, from 100:1 to 1:100 and EtOAc:CH₃OH, from 100:1 to 1:100)to afford 11-S (445 mg, 78%).

R_(f)=0.5 (CH₂Cl₂:CH₃OH, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.74 (s, 1H), 7.36 (d, J=7.9 Hz, 1H), 7.26(d, J=8.1 Hz, 1H), 7.12 (ddt, J=8.3, 7.0, 1.4 Hz, 1H), 7.02 (ddt, J=8.3,7.0, 1.4 Hz, 1H), 6.62 (s, 1H), 6.26 (d, J=1.5 Hz, 1H), 6.04 (d, J=1.5Hz, 1H), 5.12 (d, J=11.8 Hz, 1H), 4.59 (s, 1H), 4.42 (s, 1H), 4.36-4.17(m, 3H), 3.81 (s, 3H), 3.51-3.39 (m, 3H), 2.98-2.75 (m, 4H), 2.69-2.60(m, 2H), 2.47 (d, J=16.1 Hz, 1H), 2.38 (s, 3H), 2.35-2.17 (m, 2H), 2.28(s, 3H), 2.13 (s, 3H), 2.04 (s, 3H).

ESI-MS m/z: 793.3 (M+H)⁺.

C)

To a solution of 11-S (435 mg, 0.55 mmol) in CH₃CN:H₂O (1.39:1, 38.5 mL,0.015 M) was added AgNO₃ (1.84 g, 11 mmol). After 24 h at 23° C., thereaction was quenched with a mixture 1:1 of saturated aqueous solutionsof NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂, stirred for5 min, and extracted with CH₂Cl₂. The combined organic layers were driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Theresidue obtained was purified by flash chromatography (CH₂Cl₂:CH₃OH,from 99:1 to 85:15) to give 12-S (152 mg, 35%).

R_(f)=0.2 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (500 MHz, CD3OD): δ 7.34 (dd, J=7.7, 1.5 Hz, 1H), 7.28 (dd,J=7.7, 1.5 Hz, 1H), 7.04 (ddt, J=8.2, 7.0, 1.1 Hz, 1H), 6.95 (ddt,J=8.2, 7.0, 1.2 Hz, 1H), 6.55 (s, 1H), 6.31-6.25 (m, 1H), 6.15-6.05 (m,1H), 5.31 (d, J=11.4 Hz, 1H), 4.91 (s, 1H), 4.64 (s, 1H), 4.40-4.19 (m,3H), 3.76 (s, 3H), 3.64 (d, J=5.2 Hz, 1H), 3.44 (d, J=9.0 Hz, 1H),3.03-2.85 (m, 4H), 2.85-2.65 (m, 2H), 2.59 (d, J=15.6 Hz, 1H), 2.52-2.39(m, 2H), 2.37 (s, 3H), 2.27 (s, 3H), 2.09 (s, 3H), 2.00 (s, 3H).

¹³C NMR (126 MHz, CD3OD): δ 171.4, 169.3, 148.6, 145.8, 143.5, 141.2,140.8, 136.5, 131.2, 130.3, 129.5, 126.3, 121.6, 121.2, 119.8, 119.4,118.6, 117.5, 114.9, 111.0, 107.5, 107.4, 102.2, 91.1, 63.5, 60.5, 59.2,58.5, 55.3, 54.7, 53.4, 52.7, 48.6, 44.7, 42.7, 39.9, 24.3, 23.4, 19.2,15.1, 8.2.

ESI-MS m/z: 766.2 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 766.2958 [M−H₂O+H]⁺ (Calcd. for C41H₄₄N₅O₈S:766.2905).

C′)

To a solution of 10-S (5 mg, 0.006 mmol) in CH₃CN:H₂O (1.39:1, 0.5 mL,0.015 M) was added AgNO₃ (29 mg, 0.17 mmol). After 20 h at 23° C., thereaction mixture was quenched with a mixture 1:1 of saturated aqueoussolutions of NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂,stirred for 5 min, and extracted with CH₂Cl₂. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to give 13-S (5 mg, 100%).

R_(f)=0.40 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.75 (s, 1H), 7.37 (d, J=7.9 Hz, 1H),7.32-7.20 (m, 1H), 7.12 (t, J=7.7 Hz, 1H), 7.02 (t, J=7.4 Hz, 1H), 6.84(s, 1H), 6.24 (s, 1H), 6.08-5.97 (m, 1H), 6.01 (s, 1H), 5.87 (s, 1H),5.42-5.19 (m, 4H), 4.88 (s, 1H), 4.69-4.65 (m, 2H), 4.58 (s, 1H),4.28-4.13 (m, 2H), 3.84 (s, 3H), 3.68-3.40 (m, 2H), 3.24-3.15 (m, 2H),3.08-2.90 (m, 2H), 2.73-2.57 (m, 2H), 2.53-2.37 (m, 3H), 2.34 (s, 3H),2.25 (s, 3H), 2.14 (s, 3H), 2.10-2.16 (m, 1H), 2.03 (s, 3H).

ESI-MS m/z: 850.3 (M−H₂O+H)⁺.

Example 0-7 A) Synthesis of (S)-5-methoxy-tryptophanol (17-S)

To a solution of LiAlH₄ (23.4 mL, 1.0 M in THF, 23.4 mmol) at −40° C.was added carefully H₂SO₄ (0.31 mL, 5.57 mmol) and a suspension of5-methoxy-L-tryptophan (16-S) (1.0 g, 4.26 mmol, Chem-Impex) in THF(13.4 mL, 0.3 M). The reaction mixture was left evolution at 23° C.,heated for 3 h at 80° C. and 18 h at 23° C. Cool at −21° C. the reactionmixture was quenched carefully with NaOH 2N until basic pH. EtOAc wasadded and the mixture filtered through Celite® and washed with CH₃OH.The crude was concentrated under vacuum to give 17-S as a crude whichwas used in the next step without further purification.

R_(f)=0.2 (CH₂Cl₂:CH₃OH, 4:1).

¹H NMR (400 MHz, CDCl₃): δ 7.19 (dt, J=8.8, 0.7 Hz, 1H), 7.06-7.00 (m,2H), 6.72 (dd, J=8.8, 2.4 Hz, 1H), 3.77 (s, 3H), 3.63-3.48 (m, 1H),3.42-3.33 (m, 1H), 3.17-3.06 (m, 1H), 2.86 (ddt, J=14.3, 6.1, 0.8 Hz,1H), 2.66 (dd, J=14.3, 7.5 Hz, 1H).

ESI-MS m/z: 221.4 (M+H)⁺.

B) Synthesis of (R)-5-methoxy-tryptophanol (17-R)

To a solution of LiAlH₄ (11.7 mL, 1.0 M in THF, 11.7 mmol) at −40° C.was added carefully H₂SO₄ (0.31 mL, 5.75 mmol) and a suspension of5-methoxy-D-tryptophan (16-R) (0.5 g, 2.13 mmol, Aldrich) in THF (6.7mL, 0.3 M). The reaction mixture was left evolution at 23° C., heatedfor 3.5 h at 80° C. and 18 h at 23° C. Cool at −21° C. the reactionmixture was quenched carefully with NaOH 2N until basic pH. EtOAc wasadded and the mixture filtered through Celite® and washed with CH₃OH.The crude was concentrated under vacuum to give 17-R as a crude whichwas used in the next step without further purification.

R_(f)=0.2 (CH₂Cl₂:CH₃OH, 4:1).

¹H NMR (400 MHz, CD3OD): δ 7.20 (d, J=8.9 Hz, 1H), 7.06-6.96 (m, 2H),6.71 (dd, J=8.8, 2.5 Hz, 1H), 3.75 (s, 3H), 3.62-3.52 (m, 1H), 3.37 (dd,J=10.8, 7.0 Hz, 1H), 3.09 (br s, 1H), 2.82 (dd, J=14.3, 5.9 Hz, 1H),2.62 (dd, J=14.4, 7.6 Hz, 1H).

ESI-MS m/z: 221.6 (M+H)⁺.

Example 0-8 A)

To a solution of 1 (530 mg, 0.85 mmol) in acetic acid (10.6 mL, 0.08 M)was added 17-S (469 mg, 2.13 mmol). The reaction mixture was stirred at50° C. for 18 h and then acetic acid was evaporated. An aqueoussaturated solution of NaHCO₃ was added and the mixture was extractedwith CH₂Cl₂. The combined organic layers were dried over anhydrousNa₂SO₄, filtered, and concentrated under vacuum. Flash chromatography(Hexane:EtOAc, 1:1) gave compound 18-S (420 mg, 60%).

R_(f)=0.3 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CD3OD): δ 7.13 (d, J=8.8 Hz, 1H), 6.80 (d, J=2.4 Hz,1H), 6.66 (dd, J=8.8, 2.5 Hz, 1H), 6.51 (s, 1H), 6.27 (s, 1H), 6.11 (s,1H), 5.21 (d, J=11.7 Hz, 1H), 4.67 (s, 1H), 4.49-4.29 (m, 4H), 3.75 (s,3H), 3.73 (s, 3H), 3.47 (t, J=5.8 Hz, 3H), 3.37 (d, J=5.1 Hz, 1H),3.01-2.81 (m, 2H), 2.75 (d, J=7.4 Hz, 1H), 2.66 (dd, J=15.1, 4.1 Hz,1H), 2.55-2.35 (m, 4H), 2.34 (s, 3H), 2.28 (s, 3H), 2.11 (s, 3H), 1.99(s, 3H).

ESI-MS m/z: 824.3 (M+H)⁺.

B)

To a solution of 18-S (420 mg, 0.519 mmol) in CH₃CN:H₂O (1.39:1, 36 mL,0.015 M) was added AgNO₃ (2.60 g, 15.3 mmol). After 3 h at 23° C., thereaction mixture was quenched with a mixture 1:1 of saturated aqueoussolutions of NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂,stirred for 5 min, and extracted with CH₂Cl₂. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to obtain 19-S (250 mg, 60%).

R_(f)=0.45 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (500 MHz, CD3OD): δ 7.15 (dd, J=8.9, 0.6 Hz, 1H), 6.82 (dd,J=2.4, 0.6 Hz, 1H), 6.68 (dd, J=8.8, 2.5 Hz, 1H), 6.54 (s, 1H), 6.27 (d,J=1.3 Hz, 1H), 6.08 (d, J=1.3 Hz, 1H), 5.30 (d, J=11.5 Hz, 1H), 4.62 (s,1H), 4.34 (dd, J=11.4, 2.0 Hz, 1H), 4.31-4.27 (m, 2H), 3.76 (s, 3H),3.75 (s, 3H), 3.66-3.58 (m, 1H), 3.55-3.45 (m, 2H), 3.42 (d, J=7.8 Hz,1H), 2.93-2.73 (m, 3H), 2.68 (dd, J=15.1, 4.2 Hz, 1H), 2.54 (d, J=15.4Hz, 1H), 2.42 (dd, J=15.1, 10.1 Hz, 2H), 2.35 (s, 3H), 2.29 (s, 3H),2.09 (s, 3H), 2.00 (s, 3H).

¹³C NMR (126 MHz, CD3OD): δ 172.7, 170.8, 155.1, 149.9, 147.2, 145.0,142.6, 142.2, 133.1, 132.4, 132.1, 131.3, 128.1, 122.5, 121.6, 120.3,116.4, 113.0, 112.9, 111.4, 109.0, 103.6, 100.8, 92.5, 66.6, 65.0, 61.7,60.4, 59.9, 56.7, 56.1, 54.8, 54.1, 51.7, 44.1, 41.3, 30.7, 25.4, 24.7,20.6, 16.3, 9.5.

ESI-MS m/z: 798.1 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 797.2899 [M−H₂O+H]⁺ (Calcd. for C₄₂H₄₅N₄O₁₀S797.2851).

Example 0-9 A)

To a solution of 1 (311 mg, 0.50 mmol) in acetic acid (6.25 mL, 0.08 M)was added 17-R (220 mg, 1.0 mmol). The reaction mixture was stirred at50° C. for 18 h and then acetic acid was evaporated. An aqueoussaturated solution of NaHCO₃ was added and the mixture was extractedwith CH₂Cl₂. The combined organic layers were dried over anhydrousNa₂SO₄, filtered, and concentrated under vacuum. Flash chromatography(Hexane:EtOAc, 1:1) gave compound 18-R (280 mg, 68%).

R_(f)=0.3 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.53 (s, 1H), 7.18 (d, J=8.7 Hz, 1H), 6.82(d, J=2.4 Hz, 1H), 6.78 (dd, J=8.6, 2.3 Hz, 1H), 6.60 (s, 1H), 6.23 (s,1H), 6.02 (s, 1H), 5.76 (s, 1H), 5.04 (d, J=11.7 Hz, 1H), 4.62 (s, 1H),4.36 (s, 1H), 4.28 (d, J=5.0 Hz, 1H), 4.24-4.09 (m, 3H), 3.81 (s, 3H),3.79 (s, 3H), 3.64 (s, 1H), 3.47-3.40 (m, 3H), 3.01-2.90 (m, 2H), 2.53(d, J=6.9 Hz, 2H), 2.45-2.41 (m, 1H), 2.40 (s, 3H), 2.27 (s, 3H),2.22-2.14 (m, 1H), 2.18 (s, 3H), 2.06 (s, 3H).

ESI-MS m/z: 824.3 (M+H)⁺.

B)

To a solution of 18-R (330 mg, 0.40 mmol) in CH₃CN:H₂O (1.39:1, 28 mL,0.015 M) was added AgNO₃ (2.04 g, 12.0 mmol). After 3 h at 23° C., thereaction was quenched with a mixture 1:1 of saturated aqueous solutionsof NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂, stirred for5 min, and extracted with CH₂Cl₂. The combined organic layers were driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Theresidue obtained was purified by flash chromatography (CH₂Cl₂:CH₃OH,from 99:1 to 85:15) to obtain 19-R (224 mg, 69%).

R_(f)=0.44 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (500 MHz, CD3OD): δ 7.14 (dd, J=8.8, 0.5 Hz, 1H), 6.83 (d, J=2.5Hz, 1H), 6.68 (dd, J=8.8, 2.5 Hz, 1H), 6.59 (s, 1H), 6.26 (d, J=1.4 Hz,1H), 6.07 (d, J=1.4 Hz, 1H), 5.21 (d, J=11.5 Hz, 1H), 4.68-4.55 (m, 1H),4.32-4.25 (m, 2H), 4.12 (dd, J=11.5, 2.1 Hz, 1H), 3.75 (s, 3H), 3.74 (s,3H), 3.60 (d, J=5.2 Hz, 1H), 3.57-3.45 (m, 3H), 3.41 (d, J=8.8 Hz, 1H),2.97-2.83 (m, 3H), 2.73 (dd, J=15.0, 3.4 Hz, 1H), 2.69 (d, J=14.9 Hz,1H), 2.34 (s, 3H), 2.30 (s, 3H), 2.20 (dd, J=15.1, 10.4 Hz, 1H), 2.12(s, 3H), 2.11-2.08 (m, 1H), 2.05 (s, 3H).

¹³C NMR (126 MHz, CD3OD): δ 173.0, 170.8, 155.0, 149.8, 147.3, 145.0,142.8, 142.3, 133.5, 133.1, 132.2, 132.1, 131.1, 130.5, 127.8, 122.5,121.7, 120.0, 116.4, 113.5, 112.9, 111.4, 110.2, 103.5, 100.9, 92.6,66.8, 64.5, 61.3, 60.4, 60.0, 56.8, 56.1, 55.9, 54.1, 44.1, 41.3, 25.6,24.5, 20.6, 16.2, 9.6.

ESI-MS m/z: 797.4 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 797.2896 [M−H₂O+H]⁺ (Calcd. for C42H₄₅N₄O₁₀S797.2851).

Example 0-10. Synthesis of allylN—[(S)-2-amino-3-(5-methoxy-1H-indol-3-yl)propyl)]carbamate (24-S)

A)

To a solution of 17-S (6.9 g, 31.4 mmol) in CH₃CN (126 mL, 4 mL/mmol)was added di-tert-butyl dicarbonate (13.7 g, 62.8 mmol). The reactionmixture was stirred at 23° C. for 5.5 h, concentrated under vacuum.Flash chromatography (CH₂Cl₂:CH₃OH, from 99:1 to 85:15) gives 20-S (4.5g, 45%).

R_(f)=0.6 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 8.04 (s, 1H), 7.25 (d, J=8.4 Hz, 1H), 7.10(d, J=2.4 Hz, 1H), 7.03 (s, 1H), 6.87 (dd, J=8.8, 2.5 Hz, 1H), 4.83 (s,1H), 3.98 (s, 1H), 3.87 (s, 3H), 3.73-3.58 (m, 2H), 2.96 (d, J=6.6 Hz,2H), 1.42 (s, 9H).

B)

To a solution of 20-S (4.5 g, 14 mmol) in CH₂Cl₂ (84 mL, 6 mL/mmol) wasadded phthalimide (4.5 g, 30.9 mmol), triphenylphosphine (8.1 g, 30.9mmol) and the mixture was cooled at 0° C. A solution of 40% of diethylazodicarboxylate (DEAD) in CH₂Cl₂ (10.4 mL, 35 mmol) was added for 15min. The reaction was stirred at 23° C. for 18 h, concentrated undervacuum. The residue obtained was purified by flash chromatography(Hexane:EtOAc, from 99:1 to 85:15) to yield 21-S (5.8 g, 92%).

R_(f)=0.55 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 8.48 (s, 1H), 7.78 (dd, J=5.5, 3.1 Hz, 2H),7.69-7.61 (m, 2H), 7.21 (d, J=8.8 Hz, 1H), 7.06 (dd, J=18.5, 2.4 Hz,2H), 6.81 (dd, J=8.8, 2.4 Hz, 1H), 4.87 (s, 1H); 4.39 (s, 1H), 3.87 (s,3H), 3.83-3.66 (m, 2H), 2.98 (d, J=6.1 Hz, 2H), 1.20 (s, 9H).

C)

To a solution of 21-S (6.29 g, 14 mmol) in ethanol (420 mL, 30 mL/mmol)was added hydrazine monohydrate (61.1 mL, 1260 mmol). The reactionmixture was stirred at 80° C. in sealed tube for 2 h, concentrated undervacuum. Flash chromatography (CH₂Cl₂:CH₃OH, from 100:1 to 50:50) affords22-S (4.2 g, 95%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 8:2).

¹H NMR (400 MHz, CDCl₃): δ 7.22 (d, J=8.8 Hz, 1H), 7.12 (d, J=2.4 Hz,1H), 7.06 (s, 1H), 6.76 (dd, J=8.8, 2.4 Hz, 1H), 4.06-3.97 (m, 1H), 3.82(s, 3H), 3.06-2.82 (m, 4H), 1.37 (s, 9H).

D)

To a solution of 22-S (4.0 g, 12.52 mmol) in CH₃CN (125 mL, 10 mL/mmol)and DMF (12 mL, 1 mL/mmol) was added N,N-diisopropylethylamine (1.8 mL,10 mmol) and allyl chloroformate (13.3 mL, 125 mmol). The reaction wasstirred at 23° C. for 5 h. The mixture was diluted with EtOAc and NH₄Clwas added and the mixture was extracted with EtOAc. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(Hexane:EtOAc, from 100:1 to 1:100) to obtain 23-S (2.65 g, 52%).

R_(f)=0.5 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 8.11 (s, 1H), 7.28-7.20 (m, 1H), 7.04 (d,J=13.1 Hz, 2H), 6.85 (dd, J=8.9, 2.4 Hz, 1H), 5.97-5.82 (m, 1H),5.33-5.24 (m, 1H), 5.19 (dt, J=10.4, 1.3 Hz, 1H), 5.11 (s, 1H), 4.82 (s,1H), 4.55 (d, J=5.6 Hz, 2H), 4.01 (s, 1H), 3.86 (s, 3H), 3.37 (d, J=13.7Hz, 1H), 3.21 (s, 1H), 2.89 (dd, J=14.5, 7.0 Hz, 1H), 1.41 (s, 9H).

E)

To a solution of 23-S (2.60 g, 6.44 mmol) in CH₂Cl₂ (106 mL, 16.6mL/mmol) was added trifluoroacetic acid (54 mL, 8.3 mL/mmol). Thereaction mixture was stirred at 23° C. for 1.5 h, concentrated undervacuum to afford 24-S (3.9 g, 100%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CD3OD): δ 8.27 (s, 1H), 7.25 (dd, J=9.0, 2.4 Hz, 1H),7.10 (s, 1H), 6.96 (d, J=2.3 Hz, 1H), 6.87 (dd, J=9.0, 2.4 Hz, 1H), 5.81(ddt, J=16.3, 10.9, 5.7 Hz, 1H), 5.23 (dd, J=19.3, 13.6 Hz, 2H), 4.49(d, J=5.9 Hz, 2H), 3.82 (s, 3H), 3.81-3.55 (m, 1H), 3.62-3.39 (m, 2H),3.08 (qd, J=15.1, 7.3 Hz, 2H).

Example 0-11 A)

To a solution of 1 (120 mg, 0.19 mmol) in acetic acid (6 mL, 0.08 M) wasadded 24-S (117 mg, 0.35 mmol). The reaction mixture was stirred at 23°C. for 18 h and then acetic acid was evaporated. An aqueous saturatedsolution of NaHCO₃ was added and the mixture was extracted with CH₂Cl₂.The combined organic layers were dried over anhydrous Na₂SO₄, filtered,and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1)gives compound 25-S (95 mg, 54%).

R_(f)=0.4 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.64 (s, 1H), 7.14 (d, J=8.8 Hz, 1H), 6.80(s, 1H), 6.77 (d, J=8.8 Hz, 1H), 6.68 (s, 1H), 6.24 (s, 1H), 6.03 (s,1H), 6.02-5.93 (m, 1H), 5.76 (s, 1H), 5.38 (d, J=10.5 Hz, 1H), 5.26 (d,J=10.5 Hz, 1H), 5.11 (d, J=11.7 Hz, 1H), 4.66 (d, J=5.6 Hz, 2H), 4.57(s, 1H), 4.37 (s, 1H), 4.33-4.19 (m, 3H), 3.82 (s, 3H), 3.79 (s, 3H),3.46 (s, 2H), 3.17 (s, 1H), 3.10-2.90 (m, 3H), 2.68-2.45 (m, 2H),2.38-2.33 (m, 1H), 2.32 (s, 3H), 2.27 (s, 3H), 2.16 (s, 3H), 2.04 (s,2H).

ESI-MS m/z: 907.1 (M+H)⁺.

B)

To a solution of 25-S (90 mg, 0.1 mmol) in CH₂Cl₂ (2 mL, 18 mL/mmol) wasadded bis(triphenylphosphine)palladium(II)dichloride (12 mg, 0.1 mmol)and acetic acid (0.056 mL, 0.99 mmol). Tributyltin hydride (0.16 mL,0.60 mmol) was added at 0° C., the reaction mixture was stirred at 0° C.for 0.5 h, and concentrated under vacuum. Flash chromatography(Hexane:EtOAc, from 100:1 to 1:100 and EtOAc:CH₃OH, from 100:1 to 1:100)to afford 26-S (75 mg, 92%).

R_(f)=0.25 (CH₂Cl₂:CH₃OH, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.62 (s, 1H), 7.15 (d, J=9.3 Hz, 1H),6.81-6.76 (m, 2H), 6.72 (s, 1H), 6.25 (d, J=1.2 Hz, 1H), 6.03 (d, J=1.2Hz, 1H), 5.12 (d, J=11.7 Hz, 1H), 4.57 (s, 1H), 4.41 (s, 1H), 4.36-4.24(m, 2H), 4.20 (d, J=11.7 Hz, 1H), 3.82 (s, 3H), 3.79 (s, 3H), 3.44 (dd,J=22.0, 7.1 Hz, 2H), 3.08-2.78 (m, 4H), 2.73-2.64 (m, 2H), 2.41-2.22 (m,3H), 2.28 (s, 3H), 2.25-2.15 (m, 1H), 2.14 (s, 3H), 2.08 (s, 3H), 2.04(s, 3H).

ESI-MS m/z: 823.3 (M+H)⁺.

C)

To a solution of 26-S (70 mg, 0.085 mmol) in CH₃CN:H₂O (1.39:1, 6 mL,0.015 M) was added AgNO₃ (335 mg, 1.7 mmol). After 18 h at 23° C., thereaction was quenched with a mixture 1:1 of saturated aqueous solutionsof NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂, stirred for5 min, and extracted with CH₂Cl₂. The combined organic layers were driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Theresidue obtained was purified by flash chromatography (CH₂Cl₂:CH₃OH,from 99:1 to 85:15) to give 27-S (23 mg, 33%).

R_(f)=0.2 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.62 (s, 1H), 7.15 (d, J=7.8 Hz, 1H), 6.78(s, 1H), 6.75 (d, J=7.8 Hz, 1H), 6.21 (d, J=1.5 Hz, 1H), 6.01 (d, J=1.5Hz, 1H), 5.78 (s, 1H), 5.22 (d, J=11.5 Hz, 1H), 4.90 (s, 1H), 4.58-4.42(m, 3H), 4.29-4.10 (m, 2H), 3.84-3.80 (m, 1H), 3.83 (s, 3H), 3.79 (s,3H), 3.53-3.48 (m, 2H), 3.22 (d, J=8.7 Hz, 1H), 3.12 (s, 1H), 3.02 (d,J=12.8 Hz, 1H), 2.89-2.64 (m, 3H), 2.46 (s, 3H), 2.42-2.34 (m, 2H), 2.27(s, 3H), 2.12 (s, 3H), 2.03 (s, 3H).

¹³C NMR (126 MHz, CDCl₃): δ 172.1, 168.7, 154.0, 147.6, 145.6, 143.0,141.2, 140.8, 131.6, 130.6, 129.6, 127.1, 121.8, 120.9, 118.4, 115.2,112.5, 111.8, 101.8, 100.2, 81.5, 62.6, 60.6, 58.0, 57.8, 56.0, 55.8,55.0, 42.3, 41.4, 31.9, 29.7, 27.8, 26.9, 25.6, 24.0, 22.7, 20.5, 16.0,14.1, 13.6, 9.7.

ESI-MS m/z: 796.3 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 796.3062 [M−H₂O+H]⁺ (Calcd. for C42H₄₆N₅O₉S796.3011).

Example 0-12. Synthesis of allylN—[(R)-2-amino-3-(5-methoxy-1H-indol-3-yl)propyl)]carbamate (24-R)

A)

To a solution of 17-R (2.35 g, 10.7 mmol) in CH₃CN (43 mL, 4 mL/mmol)was added di-tert-butyl dicarbonate (4.67 g, 21.4 mmol). The reactionmixture was stirred at 23° C. for 2.5 h, concentrated under vacuum.Flash chromatography (CH₂Cl₂:CH₃OH, from 99:1 to 85:15) afforded 20-R(1.7 g, 50%).

R_(f)=0.6 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 8.05 (s, 1H), 7.25 (d, J=8.9 Hz, 1H), 7.09(d, J=2.4 Hz, 1H), 7.02 (d, J=2.4 Hz, 1H), 6.86 (dd, J=8.8, 2.4 Hz, 1H),4.83 (s, 1H), 3.98 (s, 1H), 3.87 (s, 3H), 3.69 (td, J=9.2, 7.5, 5.3 Hz,1H), 3.61 (dd, J=10.9, 5.6 Hz, 1H), 2.95 (d, J=6.8 Hz, 2H), 1.42 (s,9H).

B)

To a solution of 20-R (1.7 g, 5.3 mmol) in CH₂Cl₂ (32 mL, 6 mL/mmol) wasadded phthalimide (1.72 g, 11.7 mmol), triphenylphosphine (3.06 g, 11.7mmol) and the mixture was cooled at 0° C. A solution of 40% of diethylazodicarboxylate (DEAD) in CH₂Cl₂ (4.0 mL, 13.2 mmol) was added for 15min. The reaction was stirred at 23° C. for 16 h, concentrated undervacuum. The residue obtained was purified by flash chromatography(Hexane:EtOAc, from 99:1 to 85:15) to afford 21-R (2.0 g, 84%).

R_(f)=0.45 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 8.31 (s, 1H), 7.80 (dd, J=5.4, 3.0 Hz, 2H),7.67 (dd, J=5.4, 3.0 Hz, 2H), 7.30-7.12 (m, 2H), 7.08 (dd, J=15.2, 2.4Hz, 1H), 6.84 (dd, J=8.8, 2.4 Hz, 1H), 4.85 (d, J=9.2 Hz, 1H), 4.43 (q,J=5.3 Hz, 1H), 3.86 (s, 3H), 3.83-3.68 (m, 2H), 3.01 (d, J=5.4 Hz, 2H),1.22 (s, 9H).

C)

To a solution of 21-R (2.0 g, 4.45 mmol) in ethanol (133 mL, 30 mL/mmol)was added hydrazine monohydrate (21.6 mL, 445 mmol). The reactionmixture was stirred at 80° C. in sealed tube for 2 h, concentrated undervacuum. Flash chromatography (CH₂Cl₂:CH₃OH, from 100:1 to 50:50) toafford 22-R (1.15 g, 81%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 8:2).

¹H NMR (400 MHz, CDCl₃): δ 7.21 (d, J=8.8 Hz, 1H), 7.12 (s, 1H), 7.05(s, 1H), 6.75 (dd, J=8.8, 2.4 Hz, 1H), 3.95 (ddd, J=10.7, 8.7, 5.4 Hz,1H), 3.82 (s, 3H), 2.98-2.79 (m, 3H), 2.75 (dd, J=13.1, 9.4 Hz, 1H),1.37 (s, 9H).

D)

To a solution of 22-R (1.1 g, 3.4 mmol) in CH₃CN (34 mL, 10 mL/mmol) andDMF (3.4 mL, 1 mL/mmol) was added N,N-diisopropylethylamine (0.5 mL, 2.7mmol) and allyl chloroformate (3.7 mL, 34 mmol). The reaction wasstirred at 23° C. for 19 h. The mixture was diluted with EtOAc and NH₄Clwas added and the mixture was extracted with EtOAc. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(Hexane:EtOAc, from 100:1 to 1:100) to afford 23-R (0.95 g, 69%).

R_(f)=0.5 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 8.55 (s, 1H), 7.20 (d, J=8.8 Hz, 1H), 7.05(s, 1H), 6.98-6.87 (m, 1H), 6.82 (dt, J=8.8, 1.8 Hz, 1H), 5.96-5.81 (m,1H), 5.37-5.22 (m, 2H), 5.22-5.14 (m, 1H), 5.02-4.97 (m, 1H), 4.60-4.47(m, 2H), 4.00 (s, 1H), 3.84 (s, 3H), 3.31 (s, 1H), 3.19 (s, 1H), 2.88(td, J=14.5, 13.3, 5.9 Hz, 2H), 1.40 (s, 9H).

E)

To a solution of 23-R (0.94 g, 2.3 mmol) in CH₂Cl₂ (39 mL, 16.6 mL/mmol)was added trifluoroacetic acid (19 mL, 8.3 mL/mmol). The reactionmixture was stirred at 23° C. for 1.5 h, concentrated under vacuum toafford 24-R (0.72 g, 100%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CD3OD): δ 7.27 (d, J=8.8, 1H), 7.18 (s, 1H), 7.04 (d,J=2.4 Hz, 1H), 6.80 (ddd, J=8.8, 2.4, 0.9 Hz, 1H), 5.95 (ddt, J=16.4,10.8, 5.5 Hz, 1H), 5.32 (d, J=17.1 Hz, 1H), 5.20 (d, J=10.5 Hz, 1H),4.60-4.53 (m, 2H), 3.83 (s, 3H), 3.59 (dt, J=11.4, 5.5 Hz, 1H),3.47-3.30 (m, 2H), 3.13-2.94 (m, 2H).

Example 0-13 A)

To a solution of 1 (0.71 g, 1.14 mmol) in acetic acid (45 mL, 0.08 M)was added 24-R (0.54 mg, 1.8 mmol). The reaction mixture was stirred at23° C. for 7 h and then acetic acid was evaporated. An aqueous saturatedsolution of NaHCO₃ was added and the mixture was extracted with CH₂Cl₂.The combined organic layers were dried over anhydrous Na₂SO₄, filtered,and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, 1:1)gives compound 25-R (670 mg, 65%).

R_(f)=0.4 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.52 (s, 1H), 7.17 (d, J=8.8 Hz, 1H),6.83-6.73 (m, 2H), 6.61 (s, 1H), 6.23 (d, J=1.0 Hz, 1H), 6.02 (d, J=1.0Hz, 1H), 6.05-5.89 (m, 1H), 5.75 (s, 1H), 5.44-5.30 (m, 1H), 5.25 (d,J=10.4 Hz, 1H), 5.13-4.99 (m, 2H), 4.71-4.59 (m, 2H), 4.36 (s, 1H),4.30-4.07 (m, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.61-3.53 (m, 1H);3.48-3.41 (m, 3H), 3.26 (dt, J=13.3, 3.8 Hz, 1H), 3.04-2.88 (m, 2H),2.52 (dd, J=14.9, 3.7 Hz, 1H), 2.46-2.35 (m, 2H), 2.31 (s, 3H), 2.29 (s,3H), 2.16 (s, 3H), 2.12-2.02 (m, 1H), 2.09 (s, 3H).

ESI-MS m/z: 907.3 (M+H)⁺.

B)

To a solution of 25-R (745 mg, 0.82 mmol) in CH₂Cl₂ (15 mL, 18 mL/mmol)was added bis(triphenylphosphine)palladium(II) dichloride (92 mg, 0.1mmol) and acetic acid (0.47 mL, 8.2 mmol). Tributyltin hydride (1.33 mL,4.9 mmol) was added at 0° C., the reaction mixture was stirred at 0° C.for 0.75 h and concentrated under vacuum. Flash chromatography(Hexane:EtOAc, from 100:1 to 1:100 and EtOAc:CH₃OH, from 100:1 to 1:100)to afford 26-R (680 mg, >100%).

R_(f)=0.25 (CH₂Cl₂:CH₃OH, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.57 (s, 1H), 7.16 (d, J=8.8 Hz, 1H),6.85-6.72 (m, 2H), 6.57 (s, 1H), 6.21 (d, J=1.4 Hz, 1H), 6.00 (d, J=1.3Hz, 1H), 5.05-4.97 (m, 1H), 4.63 (s, 1H), 4.35 (s, 1H), 4.31-4.09 (m,4H), 3.80 (s, 3H), 3.78 (s, 3H), 3.50-3.40 (m, 3H), 3.24 (dq, J=9.9, 5.3Hz, 1H), 2.95 (s, 1H), 2.91-2.75 (m, 2H), 2.62 (dd, J=14.8, 3.6 Hz, 1H),2.43-2.28 (m, 2H), 2.36 (s, 3H), 2.25 (s, 3H), 2.22-2.14 (m, 1H), 2.15(s, 3H), 2.08 (s, 3H).

ESI-MS m/z: 823.3 (M+H)⁺.

C)

To a solution of 26-R (660 mg, 0.80 mmol) in CH₃CN:H₂O (1.39:1, 56 mL,0.015 M) was added AgNO₃ (2.70 g, 16.0 mmol). After 16.5 h at 23° C.,the reaction was quenched with a mixture 1:1 of saturated aqueoussolutions of NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂,stirred for 5 min, and extracted with CH₂Cl₂. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to give 27-R (271 mg, 42%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.46 (s, 1H), 7.16 (d, J=8.9 Hz, 1H), 6.83(s, 1H), 6.72 (d, J=8.9 Hz, 1H), 6.58 (s, 1H), 6.20 (d, J=1.8 Hz, 1H),5.99 (d, J=1.8 Hz, 1H), 5.76 (s, 1H), 5.15 (d, J=11.4 Hz, 1H), 4.86 (s,1H), 4.52 (m, 2H), 4.17 (d, J=5.3 Hz, 1H), 4.07 (d, J=11.4 Hz, 1H), 3.80(s, 3H), 3.78 (s, 3H), 3.55-3.43 (m, 2H), 3.32-3.20 (m, 2H), 3.01-2.82(m, 4H), 2.68-2.59 (m, 1H), 2.44-2.31 (m, 1H), 2.38 (s, 3H), 2.30-2.19(m, 1H), 2.26 (s, 3H), 2.15 (s, 3H), 2.07 (s, 3H).

¹³C NMR (101 MHz, CD3OD): δ 171.7, 171.3, 153.8, 153.3, 148.0, 147.6,145.4, 145.4, 143.1, 141.3, 140.7, 131.6, 131.4, 131.2, 129.3, 126.8,121.6, 120.9, 118.3, 115.6, 112.2, 111.8, 101.8, 100.2, 81.7, 63.5,63.1, 61.7, 58.0, 57.8, 56.1, 55.8, 55.0, 42.2, 42.1, 41.4, 41.0, 25.1,23.8, 20.5, 16.0, 9.7.

ESI-MS m/z: 796.3 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 796.3045 [M−H₂O+H]⁺ (Calcd. for C₄₂H₄₆N₅O₉S796.3011).

Example 0-14 A)

To a solution of compound 1 (2.0 g, 3.21 mmol) in acetonitrile (200 mL,0.01 M) was added 2-benzofuran-3-yl-ethylamine hydrochloride (30) (1.90g, 9.65 mmol, Sigma Aldrich) and cyanuric chloride (TCT) (200 mg, 10%).The reaction mixture was stirred at 85° C. for 24 h and then aqueoussaturated solution of NaHCO₃ was added and the mixture was extractedwith CH₂Cl₂. The combined organic layers were dried over anhydrousNa₂SO₄, filtered, and concentrated under vacuum. Flash chromatography(Hexane:EtOAc, from 9:1 to 1:9) gives compound 31 (1.95 g, 79%).

R_(f)=0.5 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.38-7.36 (m, 2H), 7.19-7.10 (m, 2H), 6.64(s, 1H), 6.20 (d, J=1.5 Hz, 1H), 6.05 (d, J=1.5 Hz, 1H), 5.76 (s, 1H),5.05 (d, J=11.7 Hz, 1H), 4.54 (s, 1H), 4.33-4.24 (m, 2H), 4.23-4.16 (m,2H), 3.81 (s, 3H), 3.49-3.38 (m, 2H), 3.28-3.21 (m, 1H), 3.06-2.78 (m,5H), 2.57-2.50 (m, 2H), 2.37 (s, 3H), 2.27 (s, 3H), 2.21 (m, 3H), 2.08(s, 3H).

ESI-MS m/z: 765.3 (M+H)⁺.

B)

To a solution of compound 31 (380 mg, 0.49 mmol) in CH₃CN:H₂O (1.39:1,25 mL, 0.015 M) was added AgNO₃ (1.30 g, 7.45 mmol). After 5 h at 23°C., a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO₃ wasadded, stirred for 15 min, diluted with CH₂Cl₂, stirred for 5 min, andextracted with CH₂Cl₂. The combined organic layers were dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residueobtained was purified by flash chromatography (CH₂Cl₂:CH₃OH, from 99:1to 85:15) to afford compound 32 (175 mg, 47%).

R_(f)=0.40 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.35 (ddd, J=10.7, 7.6, 1.1 Hz, 2H), 7.14(dtd, J=19.7, 7.3, 1.3 Hz, 2H), 6.65 (s, 1H), 6.16 (d, J=1.5 Hz, 1H),6.01 (d, J=1.5 Hz, 1H), 5.75 (s, 1H), 5.15 (dd, J=11.5, 1.2 Hz, 1H),4.80 (s, 1H), 4.48 (d, J=3.2 Hz, 1H), 4.44 (s, 1H), 4.20-4.06 (m, 2H),3.81 (s, 1H), 3.50 (d, J=18.8 Hz, 1H), 3.30 (ddd, J=12.6, 7.9, 5.1 Hz,1H), 3.22 (d, J=9.1 Hz, 1H), 2.99 (d, J=17.9 Hz, 1H), 2.84 (dd, J=19.2,12.0 Hz, 3H), 2.59-2.49 (m, 2H), 2.36 (s, 3H), 2.27 (s, 3H), 2.21-2.14(m, 1H), 2.18 (s, 3H), 2.06 (s, 3H).

¹³C NMR (101 MHz, CDCl₃): δ 171.2, 168.7, 154.4, 150.0, 147.9, 145.5,142.9, 140.9, 140.8, 131.3, 129.0, 127.7, 123.7, 122.2, 121.2, 120.8,118.9, 118.3, 115.5, 113.5, 111.7, 101.7, 82.1, 62.7, 61.7, 60.3, 57.8,57.4, 55.9, 55.0, 42.2, 41.3, 39.7, 38.2, 29.7, 23.7, 21.3, 20.6, 15.9,9.7.

ESI-MS m/z: 738.6 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 756.2654 [M+H]⁺ (Calcd. for C₄₀H₄₂N₃O₁₀S756.2585).

Example 0-15 A)

To a solution of 1 (500 mg, 0.80 mmol) in acetic acid (10 mL, 0.08 M)was added 2-(5-methoxybenzofuran-3-yl)-ethylamine hydrochloride (33)(Diverchim, ref: DW04590) (444 mg, 1.60 mmol). The reaction mixture wasstirred at 50° C. for 6 days and then acetic acid was evaporated. Anaqueous saturated solution of NaHCO₃ was added and the mixture wasextracted with CH₂Cl₂. The combined organic layers were dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. Flashchromatography (Hexane:EtOAc, 1:1) affords 34 (270 mg, 43%).

R_(f)=0.3 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.25 (d, J=9.1 Hz, 1H), 6.80-6.73 (m, 2H),6.63 (s, 1H), 6.18 (d, J=1.4 Hz, 1H), 6.03 (d, J=1.4 Hz, 1H), 5.78 (s,1H), 5.03 (dd, J=11.5, 1.3 Hz, 1H), 4.52 (s, 1H), 4.29 (s, 1H), 4.26(dd, J=4.7, 1.5 Hz, 1H), 4.23-4.16 (m, 2H), 3.80 (s, 3H), 3.78 (s, 3H),3.46-3.43 (m, 1H), 3.43-3.37 (m, 1H), 3.24 (s, 1H), 3.03 (d, J=18.0 Hz,1H), 2.91 (dd, J=17.9, 9.2 Hz, 1H), 2.87-2.72 (m, 2H), 2.53-2.47 (m,2H), 2.36 (s, 3H), 2.27 (s, 3H), 2.20 (s, 3H), 2.06 (s, 3H).

ESI-MS m/z: 795.8 (M+H)⁺.

B)

To a solution of 34 (345 mg, 0.43 mmol) in CH₃CN:H₂O (1.39:1, 30 mL,0.015 M) was added AgNO₃ (2.20 g, 13.0 mmol). After 3 h at 23° C., amixture 1:1 of saturated aqueous solutions of NaCl and NaHCO₃ was added,stirred for 15 min, diluted with CH₂Cl₂, stirred for 5 min, andextracted with CH₂Cl₂. The combined organic layers were dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residueobtained was purified by flash chromatography (CH₂Cl₂:CH₃OH, from 99:1to 85:15) to obtain 35 (175 mg, 51%).

R_(f)=0.35 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (500 MHz, CD3OD): δ 7.27 (d, J=9.0 Hz, 1H), 6.90 (d, J=2.6 Hz,1H), 6.80 (dd, J=9.0, 2.6 Hz, 1H), 6.57 (s, 1H), 6.23 (d, J=1.2 Hz, 1H),6.05 (d, J=1.2 Hz, 1H), 5.23 (d, J=11.5 Hz, 1H), 4.27-4.08 (m, 4H), 3.77(s, 3H), 3.75 (s, 3H), 3.63 (d, J=14.1 Hz, 2H), 3.40-3.34 (m, 2H),2.93-2.87 (m, 5H), 2.80 (d, J=15.5 Hz, 1H), 2.57-2.54 (m, 2H), 2.34 (s,3H), 2.30 (s, 3H), 2.14 (s, 3H), 2.05 (s, 3H).

¹³C NMR (126 MHz, CD3OD): δ 171.9, 170.6, 157.5, 147.0, 145.0, 142.3,141.0, 132.2, 131.1, 129.1, 122.2, 120.9, 120.2, 116.3, 115.1, 114.0,112.7, 111.4, 103.5, 102.7, 92.9, 62.0, 60.3, 59.8, 59.4, 56.5, 56.2,56.0, 54.0, 43.8, 41.2, 40.7, 30.8, 30.3, 28.7, 24.5, 21.6, 20.6, 16.2,9.6.

ESI-MS m/z: 768.6 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 768.2630 [M−H₂O+H]⁺ (Calcd. for C41H₄₂N₃O₁₀S768.2585).

Example 0-16

To a solution of LiAlH₄ (148 mL, 1.0 M in THF, 148 mmol) at −40° C. wasadded carefully H₂SO₄ (7.14 mL, 72.9 mmol) and a suspension of(S)-2-amino-3-(benzofuran-3-yl)propanoic acid (36-S) (prepared asdescribed in Tetrahedron Asymmetry 2008, 19, 500-511) (5.54 g, 26.9mmol) in THE (85 mL, 0.003 M). The reaction mixture was left evolutionat 23° C., heated at 80° C. for 3 h and 18 h at 23° C. Cool at −21° C.the reaction mixture was quenched carefully with NaOH 2N until basic pH.EtOAc was added and the mixture filtered through Celite® and washed withCH₃OH. The crude was concentrated under vacuum to afford compound 37-S(3.93 g, >100%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 4:1).

¹H NMR (400 MHz, CD3OD): δ 7.67-7.62 (m, 1H), 7.61 (s, 1H), 7.51-7.41(m, 1H), 7.34-7.18 (m, 2H), 3.69-3.48 (m, 1H), 3.44 (dd, J=10.8, 6.6 Hz,1H), 3.18 (dtd, J=7.4, 6.4, 4.6 Hz, 1H), 2.88 (ddd, J=14.4, 6.1, 1.0 Hz,1H), 2.68 (ddd, J=14.4, 7.5, 0.9 Hz, 1H).

Example 0-17

To a solution of LiAlH₄ (118 mL, 1.0 M in THF, 118 mmol) at −40° C. wasadded carefully H₂SO₄ (3.1 mL, 57.8 mmol) and a suspension of(R)-2-amino-3-(benzofuran-3-yl)propanoic acid (36-R) (prepared asdescribed in Tetrahedron Asymmetry 2008, 19, 500-511) (4.4 g, 21.4 mmol)in THE (67.4 mL, 0.003 M). The reaction mixture was left evolution at23° C., heated at 80° C. for 3 h and 18 h at 23° C. Cool at −21° C. thereaction mixture was quenched carefully with NaOH 2N until basic pH.EtOAc was added and the mixture filtered through Celite® and washed withCH₃OH. The crude was concentrated under vacuum. Flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15, Silice amine) to afford compound 37-R(2.77 g, 68%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 4:1).

¹H NMR (400 MHz, CD3OD): δ 7.63-7.52 (m, 1H), 7.56 (s, 1H), 7.46-7.33(m, 1H), 7.21 (dtd, J=19.9, 7.3, 1.3 Hz, 2H), 3.57 (dd, J=10.7, 4.6 Hz,1H), 3.42 (dd, J=10.8, 6.6 Hz, 1H), 3.15 (dtd, J=7.6, 6.3, 4.6 Hz, 1H),2.84 (ddd, J=14.4, 6.0, 1.0 Hz, 1H), 2.64 (ddd, J=14.4, 7.5, 0.9 Hz,1H).

Example 0-18 A)

To a solution of compound 1 (850 mg, 1.36 mmol) in CH₃CN (136 mL, 0.01M) was added (S)-2-amino-3-(benzofuran-3-yl)propan-1-ol (37-S) (1.30 g,6.83 mmol and cyanuric chloride (TCT) (170 mg, 20%). The reactionmixture was stirred at 85° C. for 24 h and then aqueous saturatedsolution of NaHCO₃ was added and the mixture was extracted with CH₂Cl₂.The combined organic layers were dried over anhydrous Na₂SO₄, filtered,and concentrated under vacuum. Flash chromatography (Hexane:EtOAc, from9:1 to 1:9) gives compound 38-S (750 mg, 69%).

R_(f)=0.25 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.39-7.33 (m, 1H), 7.33-7.29 (m, 1H), 7.20(ddd, J=8.3, 7.2, 1.4 Hz, 1H), 7.14 (td, J=7.4, 1.0 Hz, 1H), 6.61 (s,1H), 6.21 (d, J=1.4 Hz, 1H), 6.06 (d, J=1.4 Hz, 1H), 5.74 (s, 1H), 5.08(d, J=11.2 Hz, 1H), 4.58 (s, 1H), 4.37 (s, 1H), 4.32-4.23 (m, 2H), 4.19(d, J=2.7 Hz, 1H), 3.81 (s, 3H), 3.52-3.41 (m, 3H), 3.36-3.29 (m, 1H),3.13 (d, J=9.8 Hz, 1H), 3.00-2.81 (m, 3H), 2.57 (dd, J=15.7, 4.9 Hz,1H), 2.50 (d, J=15.2 Hz, 1H), 2.37 (s, 3H), 2.31-2.25 (m, 1H), 2.29 (s,3H), 2.16 (s, 3H), 2.10 (d, J=7.2 Hz, 1H), 2.05 (s, 3H).

ESI-MS m/z: 795.2 (M)⁺.

B)

To a solution of compound 38-S (890 mg, 1.12 mmol) in CH₃CN:H₂O (1.39:1,75 mL, 0.015 M) was added AgNO₃ (4.70 g, 28.0 mmol). After 18 h at 23°C., a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO₃ wasadded, stirred for 15 min, diluted with CH₂Cl₂, stirred for 5 min, andextracted with CH₂Cl₂. The combined organic layers were dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residueobtained was purified by flash chromatography (CH₂Cl₂:CH₃OH, from 99:1to 85:15) to afford compound 39-S (500 mg, 57%).

R_(f)=0.30 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.38-7.33 (m, 1H), 7.33-7.28 (m, 1H),7.23-7.16 (m, 1H), 7.16-7.09 (m, 1H), 6.62 (s, 1H), 6.18 (d, J=1.4 Hz,1H), 6.03 (d, J=1.4 Hz, 1H), 5.71 (s, 1H), 5.19 (d, J=11.2 Hz, 1H), 4.85(s, 1H), 4.49 (s, 2H), 4.24-4.10 (m, 3H), 3.81 (s, 3H), 3.54 (d, J=4.9Hz, 1H), 3.49 (d, J=2.3 Hz, 3H), 3.33 (t, J=10.1 Hz, 2H), 3.22 (s, 1H),2.98 (s, 1H), 2.84 (d, J=7.6 Hz, 2H), 2.62-2.53 (m, 2H), 2.37 (s, 3H),2.30-2.24 (m, 1H), 2.28 (s, 3H), 2.14 (s, 3H), 2.04 (s, 3H).

¹³C NMR (126 MHz, CDCl₃): δ 172.0, 170.7, 156.1, 150.6, 149.9, 147.1,145.0, 142.4, 142.2, 132.0, 131.4, 128.7, 125.5, 123.8, 122.6, 121.6,120.1, 116.5, 114.4, 112.3, 103.5, 92.6, 66.0, 65.1, 62.2, 60.4, 59.7,56.6, 56.1, 54.8, 54.1, 51.6, 44.0, 41.3, 38.3, 30.8, 24.8, 20.6, 16.3,9.6.

ESI-MS m/z: 768.2 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 768.2652 [M−H₂O+H]⁺ (Calcd. for C41H₄₂N₃O₁₀S768.2585)

Example 0-19 A)

To a solution of compound 1 (100 mg, 0.16 mmol) in CH₃CN (16 mL, 0.01 M)was added (R)-2-amino-3-(benzofuran-3-yl)propan-1-ol (37-R) (307 mg, 1.6mmol) and cyanuric chloride (TCT) (40 mg, 40%). The reaction mixture wasstirred at 85° C. for 44 h and then aqueous saturated solution of NaHCO₃was added and the mixture was extracted with CH₂Cl₂. The combinedorganic layers were dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. Flash chromatography (Hexane:EtOAc, from 9:1to 1:9) gives compound 38-R (95 mg, 75%).

R_(f)=0.3 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.42-7.27 (m, 2H), 7.28-7.09 (m, 2H), 6.58(s, 1H), 6.20 (d, J=1.4 Hz, 1H), 6.05 (d, J=1.4 Hz, 1H), 5.79 (s, 1H),5.00 (d, J=11.4 Hz, 1H), 4.59 (s, 1H), 4.34 (s, 1H), 4.31-4.16 (m, 4H),3.80 (s, 3H), 3.79-3.76 (m, 1H), 3.63 (s, 1H), 3.54-3.40 (m, 4H),2.99-2.87 (m, 2H), 2.68 (d, J=15.0 Hz, 1H), 2.56-2.47 (m, 1H), 2.38 (s,3H), 2.27 (s, 3H), 2.17 (s, 3H), 2.07 (s, 3H).

ESI-MS m/z: 795.2 (M+H)⁺.

B)

To a solution of compound 38-R (95 mg, 0.11 mmol) in CH₃CN:H₂O (1.39:1,11 mL, 0.015 M) was added AgNO₃ (601 mg, 3.58 mmol). After 18 h at 23°C., a mixture 1:1 of saturated aqueous solutions of NaCl and NaHCO₃ wasadded, stirred for 15 min, diluted with CH₂Cl₂, stirred for 5 min, andextracted with CH₂Cl₂. The combined organic layers were dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residueobtained was purified by flash chromatography (CH₂Cl₂:CH₃OH, from 99:1to 85:15) to afford compound 39-R (66 mg, 70%).

R_(f)=0.3 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.39-7.31 (m, 2H), 7.23-7.07 (m, 2H), 6.59(s, 1H), 6.17 (d, J=1.4 Hz, 1H), 6.01 (d, J=1.4 Hz, 1H), 5.75 (s, 1H),5.12 (dd, J=11.3, 1.2 Hz, 1H), 4.84 (s, 1H), 4.56-4.43 (m, 2H),4.19-4.07 (m, 3H), 3.79 (s, 3H), 3.83-3.74 (m, 1H), 3.66-3.51 (m, 3H),3.24 (s, 1H), 2.99-2.79 (m, 2H), 2.75-2.64 (m, 1H), 2.59-2.43 (m, 2H),2.38 (s, 3H), 2.27 (s, 3H), 2.16 (s, 3H), 2.07 (s, 3H).

¹³C NMR (101 MHz, CD3OD): δ 170.5, 169.1, 154.9, 148.9, 148.5, 145.7,143.6, 141.1, 140.8, 130.6, 129.9, 127.1, 124.1, 122.4, 122.4, 121.2,120.3, 118.7, 118.2, 115.1, 113.6, 110.9, 102.1, 91.1, 65.0, 63.3, 60.2,59.0, 58.4, 55.4, 54.5, 52.7, 52.3, 42.5, 38.7, 29.4, 23.5, 23.2, 19.1,14.8, 8.3.

ESI-MS m/z: 768.2 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 767.2628 [M−H₂O+H]⁺ (Calcd. for C41H₄₂N₃O₁₀S768.2585).

Example 0-20. Synthesis of allyl—N—[(S)-2-amino-3-(benzofuran-3-yl)propyl]carbamate (44-S)

A)

To a solution of compound 37-S (1.0 g, 5.22 mmol) in CH₃CN (21 mL, 4mL/mmol) was added di-tert-butyl dicarbonate (2.28 g, 10.4 mmol). Thereaction mixture was stirred at 23° C. for 2 h, concentrated undervacuum. Flash chromatography (CH₂Cl₂:CH₃OH, from 99:1 to 85:15) toafford compound 40-S (0.5 g, 33%).

R_(f)=0.7 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.64 (d, J=7.6 Hz, 1H), 7.49 (s, 1H), 7.46(d, J=7.6 Hz, 1H), 7.36-7.19 (m, 2H), 4.94 (s, 1H), 3.98 (s, 1H),3.71-3.56 (m, 2H), 2.93 (d, J=6.9 Hz, 2H), 1.41 (s, 9H).

B)

To a solution of compound 40-S (0.5 g, 1.71 mmol) in CH₂Cl₂ (11 mL, 6mL/mmol) was added phthalimide (0.55 g, 3.77 mmol), triphenylphosphine(0.99 g, 3.77 mmol) and the mixture was cooled at 0° C. A solution of40% of diethyl azodicarboxylate (DEAD) in CH₂Cl₂ (1.26 mL, 4.29 mmol)was added for 15 min. The reaction was stirred at 23° C. for 18 h,concentrated under vacuum. The residue obtained was purified by flashchromatography (Hexane:EtOAc, from 99:1 to 40:60) to afford compound41-S (0.68 g, 94%).

R_(f)=0.8 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.89-7.79 (m, 2H), 7.83-7.62 (m, 2H),7.65-7.55 (m, 2H), 7.49-7.42 (m, 1H), 7.33-7.20 (m, 2H), 4.83 (d, J=9.0Hz, 1H), 4.39 (ddt, J=12.1, 6.3, 2.9 Hz, 1H), 3.88-3.70 (m, 2H), 2.96(d, J=6.4 Hz, 2H), 1.24 (s, 9H).

C)

To a solution of compound 41-S (345 mg, 0.82 mmol) in ethanol (25 mL, 30mL/mmol) was added hydrazine monohydrate (3.6 mL, 73.8 mmol). Thereaction mixture was stirred at 80° C. in sealed tube for 2 h,concentrated under vacuum. Flash chromatography (CH₂Cl₂:CH₃OH, from100:1 to 50:50) to afford compound 42-S (233 mg, 98%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 8:2).

¹H NMR (400 MHz, CDCl₃): δ 7.62 (d, J=7.5 Hz, 1H), 7.49-7.42 (m, 2H),7.33-7.18 (m, 2H), 4.85 (d, J=8.8 Hz, 1H), 3.91 (s, 1H), 2.91-2.76 (m,3H), 2.67 (dd, J=13.1, 6.8 Hz, 1H), 1.25 (s, 9H).

D)

To a solution of compound 42-S (280 mg, 0.96 mmol) in CH₃CN (10 mL, 10mL/mmol) and DMF (16 mL, 1 mL/mmol) was added N,N-diisopropylethylamine(0.14 mL, 0.77 mmol) and allyl chloroformate (1.02 mL, 9.64 mmol). Thereaction was stirred at 23° C. for 2 h. The mixture was diluted withEtOAc and NH₄Cl was added and the mixture was extracted with EtOAc. Thecombined organic layers were dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. The residue obtained was purified by flashchromatography (Hexane:EtOAc, from 100:1 to 1:100) to afford compound43-S (445 mg, >100%).

R_(f)=0.5 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.60 (d, J=7.6 Hz, 1H), 7.52-7.43 (m, 2H),7.34-7.20 (m, 2H), 5.90 (ddt, J=16.4, 10.8, 5.6 Hz, 1H), 5.32-5.17 (m,2H), 4.93-4.86 (m, 1H), 4.56 (d, J=5.6 Hz, 2H), 4.08-3.98 (m, 1H),3.40-3.21 (m, 2H), 2.88 (m, 2H), 1.25 (s, 9H).

E)

To a solution of compound 43-S (160 mg, 0.43 mmol) in CH₂Cl₂ (8 mL, 16.6mL/mmol) was added trifluoroacetic acid (4 mL, 8.3 mL/mmol). Thereaction mixture was stirred at 23° C. for 1.5 h, concentrated undervacuum. Flash chromatography (CH₂Cl₂:CH₃OH, from 100:1 to 50:50) toafford compound 44-S (175 mg, >100%).

R_(f)=0.2 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CD3OD): δ 7.72 (s, 1H), 7.64 (dt, J=8.4, 0.9 Hz, 1H),7.49 (dt, J=8.4, 0.9 Hz, 1H), 7.37-7.22 (m, 2H), 5.94 (ddt, J=16.3,10.7, 5.5 Hz, 1H), 5.32 (dq, J=17.3, 1.7 Hz, 1H), 5.19 (dq, J=10.6, 1.5Hz, 1H), 4.56 (dt, J=5.7, 1.5 Hz, 2H), 3.56 (qd, J=7.0, 4.4 Hz, 1H),3.46-3.32 (m, 1H), 3.32-3.24 (m, 1H), 3.03 (dd, J=14.8, 6.9 Hz, 1H),2.91 (ddd, J=14.8, 7.1, 0.9 Hz, 1H).

Example 0-21. Synthesis of allyl—N—[(R)-2-amino-3-(benzofuran-3-yl)propyl]carbamate (44-R)

A)

To a solution of compound 37-R (2.75 g, 14.4 mmol) in CH₃CN (58 mL, 4mL/mmol) was added di-tert-butyl dicarbonate (6.27 g, 28.76 mmol). Thereaction mixture was stirred at 23° C. for 2.5 h, concentrated undervacuum. Flash chromatography (CH₂Cl₂:CH₃OH, from 99:1 to 85:15) toafford compound 40-R (3.7 g, 88%).

R_(f)=0.6 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.64 (d, J=7.6 Hz, 1H), 7.52-7.43 (m, 2H),7.35-7.20 (m, 2H), 4.85 (d, J=8.2 Hz, 1H), 4.00 (bs, 1H), 3.69 (dd,J=11.0, 4.0 Hz, 1H), 3.62 (dd, J=10.9, 5.1 Hz, 1H), 2.94 (d, J=6.9 Hz,2H), 1.42 (s, 9H).

B)

To a solution of compound 40-R (3.7 g, 12.7 mmol) in CH₂Cl₂ (76 mL, 6mL/mmol) was added phthalimide (4.1 g, 28 mmol), triphenylphosphine (7.3g, 28 mmol) and the mixture was cooled at 0° C. A solution of 40% ofdiethyl azodicarboxylate (DEAD) in CH₂Cl₂ (9.4 mL, 31.7 mmol) was addedfor 15 min. The reaction was stirred at 23° C. for 16 h, concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to afford compound 41-R (4.05 g,76%).

R_(f)=0.8 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.67-7.68 (m, 4H), 7.61 (d, J=7.5 Hz, 1H),7.58 (s, 1H), 7.46 (d, J=7.5 Hz, 1H), 7.27 (dtd, J=17.2, 7.3, 1.4 Hz,2H), 4.84 (d, J=9.0 Hz, 1H), 4.46-4.30 (m, 1H), 3.89-3.66 (m, 2H), 2.97(d, J=6.4 Hz, 2H), 1.24 (s, 9H).

C)

To a solution of compound 41-R (4.0 g, 9.5 mmol) in ethanol (285 mL, 30mL/mmol) was added hydrazine monohydrate (41.5 mL, 856 mmol). Thereaction mixture was stirred at 80° C. in sealed tube for 2 h,concentrated under vacuum. Flash chromatography (CH₂Cl₂:CH₃OH, from100:1 to 50:50) to afford compound 42-R (2.2 g, 80%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 8:2).

¹H NMR (400 MHz, CDCl₃): δ 7.60 (d, J=7.5 Hz, 1H), 7.45 (s, 1H), 7.44(d, J=7.1 Hz, 1H), 7.25 (dtd, J=18.8, 7.3, 1.3 Hz, 2H), 4.94 (d, J=8.8Hz, 1H), 3.98-3.78 (m, 1H), 2.90-2.77 (m, 2H), 2.65 (dd, J=13.1, 7.0 Hz,1H), 1.40 (s, 9H).

D)

To a solution of compound 42-R (2.2 g, 7.6 mmol) in CH₃CN (76 mL, 10mL/mmol) and DMF (7.6 mL, 1 mL/mmol) was added N,N-diisopropylethylamine(1.1 mL, 6.08 mmol) and allyl chloroformate (8.05 mL, 76 mmol). Thereaction was stirred at 23° C. for 7 h. The mixture was diluted withEtOAc and NH₄Cl was added and the mixture was extracted with EtOAc. Thecombined organic layers were dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. The residue obtained was purified by flashchromatography (Hexane:EtOAc, from 100:1 to 1:100) to afford compound43-R (2.3 g, 81%).

R_(f)=0.7 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.60 (d, J=7.5 Hz, 1H), 7.52-7.43 (m, 2H),7.34-7.20 (m, 2H), 5.90 (ddt, J=17.3, 10.8, 5.6 Hz, 1H), 5.29 (d,J=17.2, 1H), 5.20 (d, J=10.4, 1H), 5.10 (t, J=6.2 Hz, 1H), 4.86 (d,J=8.4 Hz, 1H), 4.56 (d, J=5.4, 2H), 4.08-3.97 (m, 1H), 3.36 (dt, J=10.7,4.7 Hz, 1H), 3.30-3.23 (m, 1H), 2.87 (td, J=14.8, 6.5 Hz, 2H), 1.41 (s,9H).

E)

To a solution of compound 43-R (1.32 g, 3.52 mmol) in CH₂Cl₂ (60 mL,16.6 mL/mmol) was added Trifluoroacetic acid (30 mL, 8.3 mL/mmol). Thereaction mixture was stirred at 23° C. for 1.5 h, concentrated undervacuum. Flash chromatography (CH₂Cl₂:CH₃OH, from 100:1 to 50:50) toafford compound 44-R (0.90 g, 94%).

R_(f)=0.2 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃): δ 7.75 (s, 1H), 7.69-7.61 (m, 1H), 7.54-7.46(m, 1H), 7.39-7.24 (m, 2H), 5.95 (ddt, J=16.3, 10.8, 5.5 Hz, 1H), 5.32(dd, J=17.3, 1.8 Hz, 1H), 5.24-5.16 (m, 1H), 4.57 (dt, J=5.7, 1.5 Hz,2H), 3.68 (qd, J=7.1, 4.2 Hz, 1H), 3.48 (dd, J=14.8, 4.2 Hz, 1H),3.42-3.30 (m, 1H), 3.14-2.95 (m, 2H).

Example 0-22 A)

To a solution of compound 1 (750 mg, 1.2 mmol) in CH₃CN (120 mL, 0.01 M)was added compound 44-S (1370 mg, 6 mmol) and cyanuric chloride (TCT)(184 mg, 20%). The reaction mixture was stirred at 85° C. for 23 h andthen aqueous saturated solution of NaHCO₃ was added and the mixture wasextracted with CH₂Cl₂. The combined organic layers were dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. Flashchromatography (Hexane:EtOAc, from 9:1 to 1:9) gives compound 45-S (755mg, 72%).

R_(f)=0.36 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.38-7.28 (m, 2H), 7.23-7.08 (m, 2H), 6.67(s, 1H), 6.19 (d, J=1.4 Hz, 1H), 6.09-5.95 (m, 1H), 6.04 (d, J=1.4 Hz,1H), 5.92 (s, 1H), 5.80 (s, 1H), 5.44-5.34 (m, 1H), 5.26 (dq, J=10.4,1.3 Hz, 1H), 5.08 (dd, J=11.4, 1.1 Hz, 1H), 4.70-4.63 (m, 2H), 4.56 (s,1H), 4.34 (s, 1H), 4.31-4.18 (m, 3H), 3.80 (s, 3H), 3.50-3.39 (m, 2H),3.24-3.15 (m, 1H), 3.00 (dt, J=12.2, 6.0 Hz, 2H), 2.95 (d, J=5.2 Hz,2H), 2.60 (dd, J=15.4, 4.5 Hz, 2H), 2.44 (dd, J=15.6, 5.2 Hz, 1H), 2.29(s, 3H), 2.27 (s, 3H), 2.25-2.20 (m, 1H), 2.18 (s, 3H), 2.12 (s, 1H),2.04 (s, 3H).

ESI-MS m/z: 878.2 (M+H)⁺.

B)

To a solution of compound 45-S (750 mg, 0.85 mmol) in CH₂Cl₂ (15.3 mL,18 mL/mmol) was added bis(triphenylphosphine)palladium(II) dichloride(96 mg, 0.14 mmol) and acetic acid (0.5 mL, 8.5 mmol). Tributyltinhydride (1.4 mL, 5.1 mmol) was added at 0° C., and the reaction mixturewas stirred at 0° C. for 30 minutes, and was concentrated under vacuum.Flash chromatography (Hexane:EtOAc, from 100:1 to 1:100 andCH₂Cl₂:CH₃OH, from 100:1 to 1:100) to afford compound 46-S (430 mg,64%).

R_(f)=0.3 (CH₂Cl₂:CH₃OH, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.37-7.29 (m, 2H), 7.22-7.11 (m, 2H), 6.57(s, 1H), 6.21 (d, J=1.5 Hz, 1H), 6.06 (d, J=1.5 Hz, 1H), 5.07 (d, J=11.5Hz, 1H), 4.57 (s, 1H), 4.37 (s, 1H), 4.29-4.23 (m, 2H), 4.14 (s, 1H),3.79 (s, 3H), 3.50-3.47 (m, 2H), 3.38 (d, J=8.7 Hz, 1H), 2.95-2.71 (m,4H), 2.68-2.52 (m, 2H), 2.51-2.38 (m, 1H), 2.35 (s, 3H), 2.33-2.26 (m,1H), 2.29 (s, 3H), 2.17-2.08 (m, 1H), 2.10 (s, 3H), 2.04 (s, 3H).

ESI-MS m/z: 794.3 (M+H)⁺.

C)

To a solution of compound 46-S (550 mg, 0.7 mmol) in CH₃CN:H₂O (1.39:1,49 mL, 0.015 M) was added AgNO₃ (2.4 g, 14 mmol). After 16 h at 23° C.,the reaction was quenched with a mixture 1:1 of saturated aqueoussolutions of NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂,stirred for 5 min, and extracted with CH₂Cl₂. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to give compound 47-S (53 mg, 10%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (500 MHz, CDCl₃): δ 7.36 (d, 7.9 Hz, 1H), 7.33 (d, 7.4 Hz, 1H),7.23 (t, J=7.4 Hz, 1H), 7.16 (t, J=7.4 Hz, 1H), 6.77 (s, 1H), 6.20 (s,1H), 6.04 (s, 1H), 5.92 (s, 1H), 5.20 (d, J=11.1 Hz, 1H), 4.90 (s, 1H),4.50 (s, 1H), 4.46-4.39 (m, 1H), 4.25 (d, J=11.1 Hz, 1H), 4.20 (s, 1H),3.84 (s, 3H), 3.81 (d, J=4.2 Hz, 1H), 3.58 (s, 1H), 3.40-3.14 (m, 3H),2.90 (t, J=13.0 Hz, 1H), 2.76 (m, 3H), 2.50 (s, 3H), 2.46-2.37 (m, 1H),2.32-2.26 (m, 2H), 2.30 (s, 3H), 2.15 (s, 3H), 2.04 (s, 3H).

¹³C NMR (126 MHz, CD3OD): δ 170.5, 169.2, 154.6, 149.1, 148.7, 145.7,143.5, 141.0, 140.9, 131.2, 129.6, 126.9, 124.4, 122.5, 121.4, 119.7,118.7, 115.0, 112.7, 111.0, 110.7, 102.1, 91.2, 63.5, 61.2, 59.2, 58.5,55.3, 54.7, 53.4, 52.7, 43.3, 42.5, 39.9, 36.9, 29.3, 24.1, 23.6, 19.1,15.0, 8.2.

ESI-MS m/z: 767.2 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 767.2794 [M−H₂O+H]⁺ (Calcd. for C41H₄₃N₄O₉S767.2745).

Example 0-23 A)

To a solution of compound 1 (621 mg, 1 mmol) in CH₃CN (100 mL, 0.01 M)was added compound 44-R (825 mg, 3 mmol) and cyanuric chloride (TCT)(248 mg, 40%). The reaction mixture was stirred at 85° C. for 66 h andthen aqueous saturated solution of NaHCO₃ was added and the mixture wasextracted with CH₂Cl₂. The combined organic layers were dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. Flashchromatography (Hexane:EtOAc, from 9:1 to 1:9) gives compound 45-R (530mg, 58%).

R_(f)=0.4 (Hexane:EtOAc, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.42-7.28 (m, 2H), 7.23-7.08 (m, 2H), 6.60(s, 1H), 6.20 (d, J=1.4 Hz, 1H), 6.04 (d, J=1.4 Hz, 1H), 6.01-5.92 (m,1H), 5.77 (s, 1H), 5.44-5.20 (m, 2H), 5.09 (s, 1H), 5.04-4.96 (m, 1H),4.71-4.55 (m, 2H), 4.34 (s, 1H), 4.30-4.18 (m, 3H), 3.79 (s, 3H), 3.53(dd, J=10.2, 4.4 Hz, 1H), 3.46 (m, 2H), 3.50-3.40 (m, 1H), 3.03-2.87 (m,2H), 2.67 (d, J=15.0 Hz, 1H), 2.47 (dd, J=15.6, 3.7 Hz, 1H), 2.40-2.32(m, 2H), 2.30 (s, 3H), 2.29 (s, 3H), 2.19-2.12 (m, 2H), 2.16 (s, 3H),2.09 (s, 3H).

ESI-MS m/z: 878.3 (M+H)⁺.

B)

To a solution of compound 45-R (552 mg, 0.63 mmol) in CH₂Cl₂ (11.3 mL,18 mL/mmol) was added bis(triphenylphosphine)palladium(II) dichloride(70.7 mg, 0.1 mmol) and acetic acid (0.36 mL, 6.3 mmol). Tributyltinhydride (1.02 mL, 3.8 mmol) was added at 0° C. and the reaction mixturewas stirred at 0° C. for 0.5 h, and concentrated under vacuum The crudeobtained was diluted with EtOAc, saturated aqueous solution of NH₄Cl wasadded and the mixture was extracted with EtOAc. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. Flash chromatography (Hexane:EtOAc, from 100:1 to 1:100and EtOAc:CH₃OH, from 100:1 to 1:100) to afford compound 46-R (423 mg,85%).

R_(f)=0.3 (CH₂Cl₂:CH₃OH, 1:1).

¹H NMR (400 MHz, CDCl₃): δ 7.45-7.28 (m, 2H), 7.23-7.08 (m, 2H), 6.56(s, 1H), 6.19 (d, J=1.4 Hz, 1H), 6.05 (d, J=1.4 Hz, 1H), 4.98 (d, J=11.5Hz, 1H), 4.59 (s, 1H), 4.34 (s, 1H), 4.27 (dd, J=5.1, 1.7 Hz, 1H),4.22-4.16 (m, 2H), 3.80 (s, 3H), 3.49-3.39 (m, 2H), 3.31 (dq, J=9.8,5.5, 4.5 Hz, 2H), 2.95 (s, 1H), 2.83 (d, J=5.6 Hz, 2H), 2.74-2.51 (m,3H), 2.35 (s, 3H), 2.32-2.21 (m, 2H), 2.26 (s, 3H); 2.16 (s, 3H), 2.06(s, 3H).

ESI-MS m/z: 794.3 (M+H)⁺.

C)

To a solution of compound 46-R (412 mg, 0.52 mmol) in CH₃CN:H₂O (1.39:1,36 mL, 0.015 M) was added AgNO₃ (1.76 g, 10.4 mmol). After 22 h at 23°C., the reaction was quenched with a mixture 1:1 of saturated aqueoussolutions of NaCl and NaHCO₃, stirred for 15 min, diluted with CH₂Cl₂,stirred for 5 min, and extracted with CH₂Cl₂. The combined organiclayers were dried over anhydrous Na₂SO₄, filtered, and concentratedunder vacuum. The residue obtained was purified by flash chromatography(CH₂Cl₂:CH₃OH, from 99:1 to 85:15) to give compound 47-R (175 mg, 43%).

R_(f)=0.1 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (500 MHz, CDCl₃): δ 7.34 (dd, J=11.1, 7.9 Hz, 2H), 7.22-7.07 (m,2H), 6.57 (s, 1H), 6.17 (d, J=1.2 Hz, 1H), 6.01 (d, J=1.2 Hz, 1H), 5.11(d, J=11.2 Hz, 1H), 4.84 (s, 1H), 4.53-4.47 (m, 2H), 4.21-4.07 (m, 2H),3.80 (s, 3H), 3.56 (d, J=5.1 Hz, 1H), 3.43 (s, 1H), 3.24 (d, J=9.1 Hz,1H), 2.98-2.78 (m, 4H), 2.72-2.58 (m, 2H), 2.38 (s, 3H), 2.35-2.27 (m,2H), 2.28 (s, 3H), 2.14 (s, 3H), 2.08 (s, 3H).

¹³C NMR (101 MHz, CD3OD): δ 170.6, 169.1, 155.0, 148.8, 145.6, 143.7,141.1, 140.8, 130.9, 129.7, 126.9, 124.2, 122.4, 121.1, 119.6, 118.9,118.7, 115.0, 113.2, 112.5, 111.0, 102.1, 91.3, 63.3, 60.4, 59.0, 58.4,55.3, 54.6, 52.6, 51.1, 44.9, 42.4, 39.8, 38.7, 29.4, 24.0, 23.2, 19.1,15.0, 8.3.

ESI-MS m/z: 767.2 (M−H₂O+H)⁺.

(+)-HR-ESI-TOF-MS m/z: 767.2806 [M−H₂O+H]⁺ (Calcd. for C41H₄₃N₄O₉S767.2745).

Bioactivity Example of the Payloads

The aim of this assay is to evaluate the in vitro cytostatic (ability todelay or arrest tumor cell growth) or cytotoxic (ability to kill tumorcells) activity of the samples being tested.

Cell Lines

Name No ATCC Species Tissue Characteristics A549 CCL-185 human Lung lungcarcinoma (NSCLC) HT29 HTB-38 human Colon colorectal adenocarcinomaMDA-MB- HTB-26 human Breast breast adenocarcinoma 231 PSN1 CRM-CRL-human Pancreas pancreas 3211 adenocarcinoma PC-3 CRL-1435 human Prostateprostate adenocarcinoma 22Rv1 CRL-2505 human Prostate prostate carcinoma

Evaluation of Cytotoxic Activity Using the SRB and the MTT ColorimetricAssays

A colorimetric assay, using Sulforhodamine B (SRB) reaction has beenadapted to provide a quantitative measurement of cell growth andviability (following the technique described by Skehan et al. J. Natl.Cancer Inst. 1990, 82, 1107-1112). Another colorimetric assay based on3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)reduction to a purple formazan has been also used to assess theantiproliferative activity (following the technique described by Mosmannet al. J. Immunol. Meth. 1983, 65, 55-63).

These forms of assays employ 96-well cell culture microplates followingthe standards of the American National Standards Institute and theSociety for Laboratory Automation and Screening (ANSI SLAS January 2004(R2012) Oct. 12, 2011. All the cell lines used in this study wereobtained from the American Type Culture Collection (ATCC) and derivefrom different types of human cancer.

A549, HT29, MDA-MB-231 and PSN1 cells were maintained in Dulbecco'sModified Eagle Medium (DMEM) while PC-3 and 22Rv1 cells were maintainedin Roswell Park Memorial Institute Medium (RPMI). All cell lines weresupplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100U/mL penicillin, and 100 U/mL streptomycin at 37° C., 5% CO2 and 98%humidity. For the experiments, cells were harvested from subconfluentcultures using trypsinization and resuspended in fresh medium beforecounting and plating.

A549, HT29, MDA-MB-231 and PSN1 cells were seeded in 96 well microtiterplates, at 5000 cells per well in aliquots of 150 μL, and allowed toattach to the plate surface for 18 hours (overnight) in drug freemedium. After that, one control (untreated) plate of each cell line wasfixed (as described below) and used for time zero reference value.Culture plates were then treated with test compounds (50 μL aliquots of4× stock solutions in complete culture medium plus 4% DMSO) using ten2/5 serial dilutions (concentrations ranging from 10 to 0.003 μg/mL) andtriplicate cultures (1% final concentration in DMSO). After 72 hourstreatment, the antitumor effect was measured by using the SRBmethodology: Briefly, cells were washed twice with PBS, fixed for 15 minin 1% glutaraldehyde solution at room temperature, rinsed twice in PBS,and stained in 0.4% SRB solution for 30 min at room temperature. Cellswere then rinsed several times with 1% acetic acid solution andair-dried at room temperature. SRB was then extracted in 10 mM trizmabase solution and the absorbance measured in an automatedspectrophotometric plate reader at 490 nm.

An appropriate number of PC-3 and 22Rv1 cells, to reach a final celldensity in the assay ranging from 5,000 to 15,000 cells per welldepending on the cell line, were seeded in 96-well plates and allowed tostand in culture medium for 24 h at 37° C. under 5% CO2 and 98%humidity. Then, compounds or DMSO in culture medium were added to reacha final volume of 200 μL and the intended compound concentration in arange covering ten serial 2/5 dilutions starting from 0.1 μg/mL in 1%(v/v) DMSO. At this point a set of “time zero control plates” treatedwith 1% (v/v) DMSO were processed with MTT as described below. The restof the plates were incubated during 72 h under the aforementionedenvironmental conditions. Afterwards 50 μL of a 1 mg/mL MTT solution inculture medium were added to the wells and incubated for 6-8 hours at37° C. to allow formazan crystals generation. Culture medium was thenremoved and 100 μL of neat DMSO added to each well to dissolve theformazan product into a coloured solution whose absorbance at 540 nm wasfinally measured in a PolarStar Omega microplate multilabel reader (BMGLabtech, Ortenberg, Germany).

Effects on cell growth and survival were estimated by applying the NClalgorithm (Boyd M R and Paull K D. Drug Dev. Res. 1995, 34, 91-104). Thevalues obtained in triplicate cultures were fitted by nonlinearregression to a four-parameters logistic curve by nonlinear regressionanalysis. Three reference parameters were calculated (according to theaforementioned NCl algorithm) by automatic interpolation of the curvesobtained by such fitting: GI₅₀=compound concentration that produces 50%cell growth inhibition, as compared to control cultures; TGI=total cellgrowth inhibition (cytostatic effect), as compared to control cultures,and LC₅₀=compound concentration that produces 50% net cell killingcytotoxic effect).

Tables 3-9 illustrate data on the biological activity of compounds ofthe present invention.

TABLE 3 Biological activity (Molar) Compound

3-S R₁ = CN, R₄ = —CH₂OH 3a-S R₁ = CN, R₄ = —CH₂OAc 10-S R₁ = ON, R₄ =—CH₂NHAlloc 11-S R₁ = CN, R₄ = —CH₂NH₂ 4-S R₁ = OH, R₄ = —CH₂OH 4a-S R₁= OH, R₄ = —CH₂OAc 12-S R₁ = OH, R₄ = —CH₂NH₂ 13-S R₁ = OH, R₁ =—CH₂NHAlloc MDA-MB- A549 HT29 231 PSN1 PC-3 22Rv1 GI₅₀ 3-S 4.03E-102.77E-10 4.91E-10 9.95E-10 TGI 6.17E-10 >1.26E-07 5.29E-10 1.64E-09LC₅₀ >1.26E-07 >1.26E-07 6.17E-10 >1.26E-07 GI₅₀ 3a-S 3.11E-09 2.99E-092.87E-09 2.15E-09 TGI 3.23E-09 3.23E-09 3.59E-09 3.59E-09LC₅₀ >1.20E-07 >1.20E-07 4.90E-09 1.20E-08 GI50 10-S 2.05E-08 1.14E-084.79E-09 7.64E-09 TGI 3.08E-08 1.25E-08 8.44E-09 1.25·E-08 LC₅₀7.53E-08 >1.14E-06 1.60E-08 2.39E-08 GI₅₀ 11-S 8.45E-09 3.41E-092.27E-09 3.28E-09 TGI 2.65E-08 >1.26E-07 3.41E-09 4.54E-09LC₅₀ >1.26E-07 >1.26E-07 6.43E-09 8.07E-09 GI₅₀ 4-S 1.27E-09 1.27E-091.22E-09 1.78E-09 8.08E-10 3.58E-10 TGI 1.40E-09 1.40E-09 2.55E-092.29E-09 LC₅₀ >1.27E-07 >1.27E-07 6.50E-09 3.44E-09 GI₅₀ 4a-S 3.99E-093.14E-09 3.39E-09 3.02E-09 TGI 6.17E-09 3.39E-09 5.44E-09 3.27E-09LC₅₀ >1.21E-07 >1.21E-07 1.00E-08 3.51E-09 GI₅₀ 12-S 2.04E-08 4.85E-095.23E-09 3.44E-09 TGI 5.61E-08 8.42E-09 8.42E-09 5.49E-09LC₅₀ >1.28E-07 >1.28E-07 1.53E-08 1.21E-08 GI₅₀ 13-S 1.15E-08 1.15E-081.15E-08 1.96E-08 TGI 1.61E-08 1.27E-08 1.27E-08 2.88E-08 LC₅₀2.42E-08 >1.15E-06 1.38E-08 4.61E-08

TABLE 4 Biological activity (Molar) Compound

3-R R₁ = CN, R₄ = —CH₂OH 10-R R₁ = CN, R₄ = —CH₂NHAlloc 11-R R₁ = CN, R₄= —CH₂NH₂ 4-R R₁ = OH, R₄ = —CH₂OH 12-R R₁ = OH, R₄ = —CH₂NH₂ 13-R R₁ =OH, R₄ = —CH₂NHAlloc A549 HT29 MDA-MB-231 PSN1 GI₅₀ 3-R 4.03E-102.77E-10 2.77E-10 3.90E-10 TGI 5.79E-10 >1.26E-07 5.04E-10 6.05E-10LC₅₀ >1.26E-07 >1.26E-07 1.25E-09 >1.26E-07 GI₅₀ 10-R 3.76E-09 3.08E-092.85E-09 2.62E-09 TGI 5.93E-09 >1.14E-07 4.33E-09 3.88E-09LC₅₀ >1.14E-07 >1.14E-07 7.18E-09 6.61E-09 GI₅₀ 11-R 1.77E-09 1.39E-091.01E-09 1.39 E-09 TGI 4.54E-09 >1.26E-07 1.51E-09 1.89E-09LC₅₀ >1.26E-07 >1.26E-07 2.65E-09 >1.26E-07 GI₅₀ 4-R 1.27E-09 1.26E-091.27E-09 4.59E-10 TGI 1.40E-09 1.40E-09 1.40E-09 8.54E-10LC₅₀ >1.27E-07 >1.27E-07 1.53E-09 2.55E-09 GI₅₀ 12-R 1.40E-09 5.74E-103.19E-10 4.98E-10 TGI 2.93E-09 1.10E-09 6.76E-10 1.22E-09 LC₅₀ 1.22E-082.93E-09 1.40E-09 >1.28E-07 GI₅₀ 13-R 7.26E-09 6.91E-09 4.95E-092.88E-09 TGI 7.72E-09 7.60E-09 7.95E-09 3.11E-09LC₅₀ >1.15E-07 >1.15E-07 1.38E-08 3.46E-09

TABLE 5 Biological activity (Molar) Compound

38-S R₁ = CN, R₄ = —CH₂OH 45-S R₁ = CN, R₄ = —CH₂NHAlloc 46-S R₁ = CN,R₄ = —CH₂NH₂ 39-S R₁ = OH, R₄ = —CH₂OH 47-S R₁ = OH, R₄ = —CH₂NH₂MDA-MB- A549 HT29 231 PSN1 PC-3 22Rv1 GI₅₀ 38-S 8.05E-09 4.53E-092.52E-09 5.03E-09 TGI 8.55E-09 7.05E-09 4.28E-09 8.18E-09 LC₅₀9.44E-09 >1.26E-07 7.80E-09 1.51E-08 GI₅₀ 45-S 1.82E-08 1.82E-081.71E-08 1.94E-08 TGI 1.94E-08 1.94E-08 2.16E-08 2.62E-08 LC₅₀2.16E-08 >1.14E-07 2.96E-08 3.64E-08 GI₅₀ 46-S 8.19E-09 2.77E-093.65E-09 3.15E-09 TGI 2.14E-08 6.17E-09 6.80E-09 4.79E-09LC₅₀ >1.26E-07 >1.26E-07 1.26E-08 9.20E-09 GI₅₀ 39-S 4.84E-09 3.94E-093.44E-09 8.02E-09 2.78E-09 4.81E-10 TGI 8.27E-09 6.74E-09 7.13E-091.02E-08 LC₅₀ 1.65E-08 >1.27E-07 1.78E-08 1.27E-08 GI₅₀ 47-S 1.40E-084.33E-09 6.24E-09 5.99E-09 TGI 2.80E-08 6.75E-09 9.68E-09 8.54E-09LC₅₀ >1.27E-07 >1.27E-07 1.66E-08 1.27E-08

TABLE 6 Biological (Molar) activity Compound

38-R R₁ = CN, R₄ = —CH₂OH 45-R R₁ = CN, R₄ = —CH₂NHAlloc 46-R R₁ = CN,R₄ = —CH₂NH₂ 39-R R₁ = OH, R₄ = —CH₂OH 47-R R₁ = OH, R₄ = —CH₂NH₂ A549HT29 MDA-MB-231 PSN1 GI₅₀ 38-R 6.54E-10 5.41E-10 4.53E-10 6.54E-10 TGI1.04E-09 5.91E-10 8.43E-10 9.94E-10 LC₅₀ >1.26E-07 >1.26E-07 2.01E-091.76E-09 GI₅₀ 45-R 1.82E-08 1.25E-08 9.57E-09 1.06E-08 TGI 1.94E-082.28E-08 1.94E-08 1.94E-08 LC₅₀ 2.39E-08 >1.14E-07 4.33E-08 3.76E-08GI₅₀ 46-R 1.51E-09 1.21E-09 1.23E-09 9.95E-10 TGI 2.77E-09 1.39E-091.39E-09 1.51E-09 LC₅₀ >1.26E-07 >1.26E-07 1.51E-09 2.65E-09 GI₅₀ 39-R2.67E-10 2.93E-10 2.04E-10 3.65E-10 TGI 4.33E-10 6.24E-10 5.98E-105.73E-10 LC₅₀ >1.27E-07 >1.27E-07 2.80E-09 1.06E-09 GI₅₀ 47-R 2.04E-098.03E-10 5.99E-10 1.40E-09 TGI 3.82E-09 1.40E-09 1.17E-09 2.04E-09 LC₅₀1.40E-08 >1.27E-07 2.55E-09 3.31E-09

TABLE 7 Biological activity (Molar) Compound

18-S R₁ = CN, R₄ = —CH₂OH 25-S R₁ = CN, R₄ = —CH₂NHAlloc 26-S R₁ = CN,R₄ = —CH₂NH₂ 19-S R₁ = OH, R₄ = —CH₂OH 27-S R₁ = OH, R₄ = —CH₂NH₂ A549HT29 MDA-MB-231 PSN1 GI₅₀ 18-S 1.70E-09 1.21E-09 1.21E-09 9.59E-10 TGI3.03E-09 1.34E-09 1.34E-09 1.34E-09 LC₅₀ >1.21E-07 >1.21E-071.58E-09 >1.21E-07 GI₅₀ 25-S 7.17E-09 7.17E-09 5.84E-09 6.84E-09 TGI7.61E-09 7.72E-09 9.04E-09 9.26E-09 LC₅₀ >1.10E-07 >1.10E-07 1.54E-081.43E-08 GI₅₀ 26-S 1.12E-08 2.79E-09 1.34E-09 3.04E-09 TGI 2.19E-083.16E-09 1.94E-09 3.28E-09 LC₅₀ >1.22E-07 >1.22E-07 3.89E-09 3.52E-09GI₅₀ 19-S 3.07E-09 1.35E-09 1.96E-09 2.95E-09 TGI 3.31E-09 1.60E-093.31E-09 3.19E-09 LC₅₀ >1.23E-07 >1.23E-07 1.10E-08 >1.23E-07 GI₅₀ 27-S6.02E-09 1.23E-09 1.19E-09 1.97E-09 TGI 1.12E-08 1.35E-09 1.23E-092.83E-09 LC₅₀ >1.23E-07 >1.23E-07 1.35E-09 4.55E-09

TABLE 8 Biological activity (Molar) Compound

18-R R₁ = CN, R₄ = —CH₂OH 25-R R₁ = CN, R₄ = —CH₂NHAlloc 26-R R₁ = CN,R₄ = —CH₂NH₂ 19-R R₁ = OH, R₄ = —CH₂OH 27-R R₁ = OH, R₄ = —CH₂NH₂MDA-MB- A549 HT29 231 PSN1 GI₅₀ 18-R 1.21E-09 1.21E-09 1.21E-09 5.70E-10TGI 1.34E-09 1.34E-09 1.34E-09 1.06E-09 LC₅₀ >1.21E-07 >1.21E-071.46E-09 >1.21E-07 GI₅₀ 25-R 1.32E-09 1.54E-09 1.21E-09 1.21E-09 TGI2.43E-09 2.76E-09 2.54E-09 2.32E-09 LC₅₀ 9.92E-09 >1.10E-07 8.38E-096.73E-09 GI₅₀ 26-R 1.94E-09 7.29E-10 1.17E-09 9.72E-10 TGI 3.40E-091.58E-09 1.22E-09 1.70E-09 LC₅₀ >1.22E-07 >1.22E-07 1.46E-09 3.52E-09GI₅₀ 19-R 1.47E-09 1.72E-09 1.23E-09 1.23E-09 TGI 3.56E-09 1.72E-091.35E-09 1.35E-09 LC₅₀ >1.23E-07 >1.23E-07 1.23E-07 1.47E-09 GI₅₀ 27-R2.09E-09 5.04E-10 3.07E-10 6.39E-10 TGI 3.93E-09 5.53E-10 5.41E-101.17E-09 LC₅₀ 1.01E-08 >1.23E-07 8.60E-10 2.46E-09

TABLE 9 Biological activity (Molar) Compound

31 R₁ = CN, R₃ = H 32 R₁ = OH, R₃ = H 34 R₁ = CN, R₃ = OMe 35 R₁ = OH,R₃ = OMe A549 HT29 MDA-MB-231 PSN1 GI₅₀ 31 1.96E-08 1.05E-08 8.89E-096.80E-09 TGI 2.09E-08 1.57E-08 1.70E-08 1.57E-08 LC₅₀ 2.35E-08 >1.31E-073.53E-08 4.31E-08 GI₅₀ 32 6.88E-09 6.88E-09 4.76E-09 6.09E-09TGI >1.32E-08 >1.32E-08 1.05E-08 8.34E-09LC₅₀ >1.32E-08 >1.32E-08 >1.32E-08 1.20E-08 GI₅₀ 34 5.91E-08 5.41E-084.53E-08 5.41E-08 TGI 8.05E-08 8.55E-08 7.67E-08 5.91E-08 LC₅₀ >1.26E-071.25E-07 1.12E-07 >1.26E-07 GI₅₀ 35 8.14E-09 7.89E-09 4.58E-09 6.24E-09TGI 8.78E-09 8.65E-09 8.27E-09 9.03E-09 LC₅₀ >1.27E-07 >1.27E-071.65E-08 1.40E-08This data demonstrates that the payloads employed in the presentinvention have high potency in vitro.

Synthesis of Linkers Preparation of LIN 1: MC-Val-Cit-PABC-PNP

(a) Preparation of LIN 1-1: MC-Val-Cit-OH

Cl-TrtCl-resin (20 g, 1.49 mmol/g) (Iris Biotech, Ref.: BR-1065,2-Chlorotrityl chloride resin (200-400 mesh, 1% DVB, 1.0-1.6 mmol/g),CAS 42074-68-0) was placed in a filter plate. 100 mL of DCM was added tothe resin and the mixture was stirred for 1 h. The solvent waseliminated by filtration under vacuum. A solution of Fmoc-Cit-OH (11.83g, 29.78 mmol) and DIPEA (17.15 mL, 98.45 mmol) in DCM (80 mL) was addedand the mixture was stirred for 10 min. After that DIPEA (34.82 mmol,199.98 mmol) was added and the mixture was stirred for 1 h. The reactionwas terminated by addition of MeOH (30 mL) after stirring for 15minutes. The Fmoc-Cit-O-TrtCl-resin produced as a result was subjectedto the following washing/treatments: DCM (5×50 mL×0.5 min), DMF (5×50mL×0.5 min), piperidine:DMF (1:4, 1×1 min, 2×10 min), DMF (5×50 mL×0.5min), DCM (5×50 mL×0.5 min). The final piperidine wash gaveNH₂—Cit-O-TrtCl-resin. The loading was calculated: 1.15 mmol/g.

The NH₂-Cit-O-TrtCl-resin produced above was washed with DMF (5×50mL×0.5 min) and a solution of Fmoc-Val-OH (31.22 g, 91.98 mmol), HOBt(11.23 g, 91.98 mmol) in DMF (100 mL) was added to theNH₂-Cit-O-TrtCl-resin, stirred and DIPCDI (14.24 mL, 91.98 mmol) wasadded and the mixture was stirred for 1.5 h. The reaction was terminatedby washing with DMF (5×50 mL×0.5 min). The Fmoc-Val-Cit-O-TrtCl-resinthus produced was treated with piperidine:DMF (1:4, 1×1 min, 2×10 min)and washed with DMF (5×50 mL×0.5 min). The final piperidine wash gaveNH₂—Val-Cit-O-TrtCl-resin.

A solution of 6-maleimidocaproic acid (MC-OH) (9.7 g, 45.92 mmol), HOBt(6.21 g, 45.92 mmol) in DMF (100 mL) was added to theNH₂-Val-Cit-O-TrtCl-resin produced above, stirred and DIPCDI (7.12 mL,45.92 mmol) was added and the mixture was stirred for 1.5 h. Thereaction was terminated by washing with DMF (5×50 mL×0.5 min) and DCM(5×50 mL×0.5 min).

The peptide was cleaved from the resin by treatments with TFA:DCM (1:99,5×100 mL). The resin was washed with DCM (7×50 mL×0.5 min). The combinedfiltrates were evaporated to dryness under reduced pressure and thesolid obtained was triturated with Et₂O and filtrated to obtain LIN 1-1(7.60 g, 71%) as a white solid.

¹H NMR (500 MHz, DMSO-d₆): δ 12.47 (s, 1H), 8.13 (d, J=7.3 Hz, 1H), 7.74(d, J=9.0 Hz, 1H), 6.99 (s, 2H), 5.93 (s, 1H), 5.35 (s, 2H), 4.20 (dd,J=9.0, 6.8 Hz, 1H), 4.15-4.07 (m, 1H), 3.36 (t, J=7.0 Hz, 2H), 3.00-2.88(m, 2H), 2.21-2.12 (m, 1H), 2.11-2.03 (m, 1H), 1.98-1.86 (m, 1H),1.74-1.62 (m, 1H), 1.61-1.50 (m, 1H), 1.50-1.31 (m, 6H), 1.21-1.11 (m,2H), 0.84 (d, J=6.8 Hz, 3H), 0.80 (d, J=6.8 Hz, 3H).

ESI-MS m/z: Calcd. for C₂₁H₃₃N₅O₇: 467.2. Found: 468.3 (M+H)⁺.

(b) Preparation of LIN 1-2: MC-Val-Cit-PABOH

To a solution of LIN 1-1 (1.6 g, 3.42 mmol) and 4-aminobenzyl alcohol(PABOH) (0.84 g, 6.84 mmol) in DCM (60 mL) was added a solution of HOBt(0.92 g, 6.84 mmol) in DMF (5 mL). DIPCDI (1.05 mL, 6.84 mmol) wasadded, the reaction mixture was stirred for 2 h at 23° C., Et₂O (150 mL)was added, and the solid obtained was filtrated in a filter plate undervacuum to obtain LIN 1-2 (1.31 g, 67%).

¹H NMR (500 MHz, DMSO-d₆): δ 9.88 (s, 1H), 8.03 (d, J=7.6 Hz, 1H), 7.77(dd, J=12.2, 8.5 Hz, 1H), 7.53 (d, J=8.2 Hz, 2H), 7.21 (d, J=8.2 Hz,2H), 6.99 (s, 3H), 6.01-5.92 (m, 1H), 5.39 (s, 2H), 5.07 (s, 1H), 4.41(s, 2H), 4.39-4.31 (m, 1H), 4.23-4.12 (m, 1H), 3.36 (t, J=7.0 Hz, 2H),3.06-2.97 (m, 1H), 2.96-2.90 (m, 1H), 2.22-2.03 (m, 2H), 2.01-1.88 (m,1H), 1.76-1.62 (m, 1H), 1.63-1.28 (m, 6H), 1.25-1.11 (m, 2H), 0.84 (d,J=6.9 Hz, 3H), 0.81 (d, J=6.8 Hz, 3H).

ESI-MS m/z: Calcd. for C₂₈H₄₀N₆O₇: 572.3. Found: 573.3 (M+H)⁺.

(c) Preparation of LIN 1: MC-Val-Cit-PAB-PNP

To a solution of LIN 1-2 (500 mg, 0.87 mmol) and bis(4-nitrophenyl)carbonate (bis-PNP) (2.64 g, 8.72 mmol) in DCM:DMF (8:2, 25 mL) wasadded DIPEA (0.45 mL, 2.61 mmol). The reaction mixture was stirred for20 h at 23° C. and poured onto a silica gel column (DCM:CH₃OH, from 50:1to 10:1) to afford pure target LIN 1 (364 mg, 57%).

R_(f)=0.40 (CH₂Cl₂:CH₃OH, 9:1).

¹H NMR (400 MHz, CDCl₃/CD3OD): δ 9.45 (s, 1H), 8.23 (d, J=8.3 Hz, 2H),7.59 (d, J=8.5 Hz, 2H), 7.35 (d, J=8.3 Hz, 2H), 7.34 (d, J=8.5 Hz, 2H),6.65 (s, 2H), 5.20 (s, 2H), 4.56 (dt, J=10.5, 5.4 Hz, 1H), 4.15 (d,J=7.2 Hz, 1H), 3.46 (dd, J=8.0, 6.4 Hz, 2H), 3.16-2.89 (m, 2H), 2.21(dd, J=8.3, 6.6 Hz, 2H), 2.06-1.97 (m, 1H), 1.90-1.83 (m, 1H), 1.73-1.46(m, 7H), 1.34-1.20 (m, 2H), 0.91 (d, J=6.7 Hz, 3H), 0.90 (d, J=6.7 Hz,3H).

¹³C NMR (125 MHz, CDCl₃/CD3OD) δ 174.4, 172.4, 171.1, 170.6, 160.5,155.5, 152.5, 145.3, 138.7, 134.1, 129.9, 129.5, 125.2, 121.8, 120.0,70.6, 59.0, 53.2, 37.5, 35.8, 30.6, 29.6, 29.3, 28.1, 26.2, 26.2, 25.1,19.1, 18.1.

ESI-MS m/z: Calcd. for C35H₄₃N7011: 737.3. Found: 738.3 (M+H)⁺.

Preparation of LIN-2: MC2-PEG4-Val-Cit-PABC-PNP

a) Preparation of LIN 2-1: MC2-PEG4-Val-Cit-OH

Cl-TrtCl-resin (5 g, 1.49 mmol/g) was placed in a filter plate. To theresin was added CH₂Cl₂ (25 mL) and the mixture was stirred for 1 h at23° C. The solvent was eliminated by filtration over vacuum. A solutionof Fmoc-Cit-OH (2.95 g, 7.44 mmol) and DIPEA (4.29 mL, 24.61 mmol) inCH₂Cl₂ (20 mL) was added and the mixture was stirred for 10 min at 23°C. DIPEA (8.70 mL, 49.99 mmol) was additionally added and the mixturewas stirred for 1 h at 23° C. The reaction was stopped by addition ofMeOH (10 mL) and stirred 15 min at 23° C. The Fmoc-Cit-O-TrtCl-resin wassubjected to the following washing/treatments: CH₂Cl₂ (5×15 mL×0.5 min),DMF (5×15 mL×0.5 min), piperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min),DMF (5×15 mL×0.5 min), CH₂Cl₂ (5×15 mL×0.5 min). The loading wascalculated: 1.17 mmol/g.

The NH₂—Cit-O-TrtCl-resin was washed with DMF (5×15 mL×0.5 min) and asolution of Fmoc-Val-OH (7.80 g, 22.99 mmol) and HOBt (2.80 g, 24.5mmol) in DMF (25 mL) was added to the NH₂-Cit-O-TrtCl-resin followed byaddition of DIPCDI (3.56 mL, 24.5 mmol) at 23° C. The reaction mixturewas stirred for 1.5 h at 23° C. The reaction was stopped by washing withDMF (5×15 mL×0.5 min). The Fmoc-Val-Cit-O-TrtCl-resin was treated withpiperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min) and washed with DMF (5×15mL×0.5 min).

A solution of15-(9-Fluorenylmethyloxycarbonyl)amino-4,7,10,13-tetraoxa-pentadecanoicacid (Fmoc-NH-PEG4-OH) (4.27 g, 8.75 mmol) and HOBt (1.18 g, 8.72 mmol)in DMF (30 mL) was added to the NH₂-Val-Cit-O-TrtCl-resin followed byaddition of DIPCDI (1.35 mL, 8.72 mmol) at 23° C. The reaction mixturewas stirred for 24 h at 23° C. The reaction was stopped by washing withDMF (5×15 mL×0.5 min). The Fmoc-NH-PEG4-Val-Cit-O-TrtCl-resin wastreated with piperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min) and washedwith DMF (5×15 mL×0.5 min).

A solution of 3-(Maleimido)propionic acid (MC2-OH) (3.95 g, 23.35 mmol)and HOBt (3.16 g, 23.37 mmol) in DMF (30 mL) was added to theNH₂-PEG4-Val-Cit-O-TrtCl-resin followed by addition of DIPCDI (3.62 mL,23.37 mmol) at 23° C. The reaction mixture was stirred for 2 h at 23° C.The reaction was stopped by washing with DMF (5×15 mL×0.5 min) andCH₂Cl₂ (5 x 15 mL×0.5 min).

The peptide was cleaved from the resin by treatments with TFA:CH₂Cl₂(1:99, 5×50 mL). The resin was washed with CH₂Cl₂ (7×50 mL×0.5 min). Thecombined filtrates were evaporated to dryness under reduced pressure,the solid obtained was triturated with Et₂O and filtrated to obtain LIN2-1 (4.59 g, 87% yield) as a white solid.

¹H NMR (300 MHz, CDCl₃): δ 7.67-7.57 (m, 1H), 7.44 (d, J=8.3 Hz, 1H),7.11 (t, J=5.4 Hz, 1H), 6.73 (s, 2H), 4.49 (d, J=7.2 Hz, 1H), 4.35 (t,J=7.7 Hz, 1H), 3.82 (t, J=7.0 Hz, 2H), 3.74 (t, J=6.2 Hz, 2H), 3.68-3.56(m, 13H), 3.56-3.45 (m, 2H), 3.39 (q, J=5.4 Hz, 2H), 3.17 (s, 2H), 2.55(q, J=7.0, 6.0 Hz, 4H), 2.16-1.99 (m, 1H), 1.91 (s, 1H), 1.75 (s, 1H),1.43 (s, 2H), 0.94 (d, =9.7 Hz, 3H), 0.93 (d, =9.7 Hz, 3H).

ESI-MS m/z: 673.3 (M+H)⁺.

(b) Preparation of LIN 2-2: MC2-PEG4-Val-Cit-PABOH

To a solution of LIN 2-1 (1.5 g, 2.22 mmol) and 4-aminobenzyl alcohol(PABOH) (0.55 g, 4.45 mmol) in CH₂Cl₂ (60 mL) was added a solution ofHOBt (0.60 g, 4.45 mmol) in DMF (5 mL) followed by addition of DIPCDI(0.69 mL, 4.45 mmol) at 23° C. The reaction mixture was stirred for 5 hat 23° C., Et₂O (150 mL) was added, and the solid obtained was filtratedunder vacuum to obtain crude LIN 2-2 (2.37 g, >100% yield) which wasused in the next step without further purification.

¹H NMR (500 MHz, DMSO-d₆): δ 7.57 (d, J=8.6 Hz, 2H), 7.30 (d, J=8.6 Hz,2H), 6.81 (s, 2H), 4.58 (s, 1H), 4.56 (s, 2H), 4.50 (dd, J=9.1, 5.1 Hz,1H), 4.21 (d, J=7.0 Hz, 1H), 3.80-3.68 (m, 4H), 3.65-3.59 (m, 12H),3.55-3.47 (m, 1H), 3.20 (dd, J=13.6, 6.9 Hz, 1H), 3.12 (dt, J=13.5, 6.7Hz, 1H), 2.55 (td, J=6.1, 2.1 Hz, 2H), 2.46 (t, J=6.9 Hz, 2H), 2.15-2.07(m, 1H), 1.95-1.88 (m, 1H), 1.79-1.70 (m, 1H), 1.67-1.50 (m, 2H), 0.99(d, J=7.0 Hz, 3H), 0.98 (d, J=7.0 Hz, 3H).

ESI-MS m/z: 778.4 (M+H)⁺.

(c) Preparation of LIN 2: MC2-PEG4-Val-Cit-PABC-PNP

To a solution of LIN 2-2 (1.73 g, 2.22 mmol) and bis(4-nitrophenyl)carbonate (bis-PNP) (3.38 g, 11.12 mmol) in DCM:DMF (8:2, 75 mL) wasadded DIPEA (1.16 mL, 6.07 mmol) at 23° C. The reaction mixture wasstirred for 19 h at 23° C. and poured onto silica gel column(CH₂Cl₂:CH₃OH, from 50:1 to 10:1) to afford pure LIN 2 (945 mg, 45%yield).

¹H NMR (500 MHz, CD3OD): δ 8.22 (d, J=9.2 Hz, 2H), 7.61 (d, J=8.6 Hz,2H), 7.34 (d, J=9.2 Hz, 2H), 7.33 (d, J=8.6 Hz, 2H), 6.67 (s, 2H),4.57-4.47 (m, 1H), 4.23-4.12 (m, 1H), 3.78-3.76 (m, 12H), 3.63-3.50 (m,16H), 3.49-3.41 (m, 2H), 3.34-3.25 (m, 2H), 3.18-3.03 (m, 2H), 2.51 (t,J=5.9 Hz, 2H), 2.45 (t, J=7.2 Hz, 2H), 2.13-1.99 (m, 1H), 1.92-1.84 (m,1H), 1.73-1.62 (m, 1H), 1.55-1.45 (m, 2H), 0.92 (d, J=6.8 Hz, 3H), 0.90(d, J=6.8 Hz, 3H).

¹³C NMR (75 MHz, CDCl₃/CD3OD): δ 174.4, 172.9, 172.4, 172.4, 171.6,170.9, 170.8, 170.7, 163.7, 155.8, 155.7, 152.5, 145.4, 138.8, 134.1,131.3, 130.4, 129.2, 128.7, 125.7, 124.9, 121.8, 119.8 (×2), 115.1, 70.2(×2), 70.1 (×2), 70.0, 69.9, 69.8, 69.0, 66.9, 59.2, 53.5, 39.0, 36.0,34.4, 34.1, 30.4, 29.0, 18.5, 17.5.

ESI-MS m/z: 943.4 (M+H)⁺.

R_(f)=0.20 (CH₂Cl₂:CH₃OH, 9:1).

Preparation of LIN 3: MC2-PEG4-Val-Ala-PABC-PNP

(a) Preparation of LIN 3-1: MC2-PEG4-Val-Ala-OH

Cl-TrtCl-resin (5 g, 1.49 mmol/g) was placed in a filter plate. To theresin was added CH₂Cl₂ (25 mL) and the mixture was stirred for 1 h at23° C. The solvent was eliminated by filtration over vacuum. A solutionof Fmoc-Ala-OH (2.31 g, 7.41 mmol) and DIPEA (4.28 mL, 24.61 mmol) inCH₂Cl₂ (20 mL) was added and the mixture was stirred for 10 min at 23°C. DIPEA (8.60 mL, 49.37 mmol) was additionally added and the reactionmixture was stirred for 1 h at 23° C. The reaction was stopped byaddition of MeOH (10 mL) and stirred 15 min at 23° C. TheFmoc-Ala-O-TrtCl-resin was subjected to the followingwashing/treatments: CH₂Cl₂ (5 x 15 mL×0.5 min), DMF (5×15 mL×0.5 min),piperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min), DMF (5×15 mL×0.5 min),CH₂Cl₂ (5×15 mL×0.5 min). The loading was calculated: 1.34 mmol/g.

The NH₂-Ala-O-TrtCl-resin was washed with DMF (5×15 mL×0.5 min) and asolution of Fmoc-Val-OH (9.09 g, 26.79 mmol) and HOBt (3.62 g, 26.79mmol) in DMF (25 mL) was added to the NH₂-Ala-O-TrtCl-resin followed byaddition DIPCDI (4.14 mL, 26.79 mmol) at 23° C. The mixture was stirredfor 1.5 h at 23° C. The reaction was stopped by washing with DMF (5×15mL×0.5 min). The Fmoc-Val-Ala-O-TrtCl-resin was treated withpiperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min) and washed with DMF (5×15mL×0.5 min).

A solution of15-(9-Fluorenylmethyloxycarbonyl)amino-4,7,10,13-tetraoxa-pentadecanoicacid (Fmoc-NH-PEG4-OH) (4.90 g, 8.75 mmol) and HOBt (1.35 g, 9.98 mmol)in DMF (30 mL) was added to the NH₂-Val-Ala-O-TrtCl-resin followed byaddition DIPCDI (1.55 mL, 10.0 mmol) at 23° C. The reaction mixture wasstirred for 22 h at 23° C. The reaction was stopped by washing with DMF(5×15 mL×0.5 min). The Fmoc-NH-PEG4-Val-Ala-O-TrtCl-resin was treatedwith piperidine:DMF (1:4, 15 mL, 1×1 min, 2×10 min) and washed with DMF(5×15 mL×0.5 min).

A solution of 3-(Maleimido)propionic acid (MC2-OH) (4.53 g, 26.78 mmol)and HOBt (3.62 g, 26.77 mmol) in DMF (30 mL) was added to theNH₂-PEG4-Val-Ala-O-TrtCl-resin followed by addition of DIPCDI (4.15 mL,26.80 mmol) at 23° C. The reaction mixture was stirred for 2 h at 23° C.The reaction was stopped by washing with DMF (5×15 mL×0.5 min) andCH₂Cl₂ (5 x 15 mL×0.5 min).

The peptide was cleaved from the resin by treatments with TFA:CH₂Cl₂(1:99, 5×50 mL). The resin was washed with CH₂Cl₂ (7×50 mL×0.5 min). Thecombined filtrates were evaporated to dryness under reduced pressure,the solid obtained was triturated with Et₂O and filtrated to obtain L3-1 (4.73 g, 87% yield) as a white solid.

¹H NMR (500 MHz, CDCl₃): δ 7.67 (bs, 1H), 7.31 (d, J=8.9 Hz, 1H), 7.17(d, J=7.0 Hz, 1H), 6.85 (t, J=5.6 Hz, 1H), 6.72 (s, 2H), 4.51 (q, J=7.1Hz, 1H), 4.38 (dd, J=8.9, 6.9 Hz, 1H), 3.84 (t, J=7.1 Hz, 2H), 3.75 (t,J=5.9 Hz, 2H), 3.69-3.59 (m, 12H), 3.55 (t, J=5.1 Hz, 2H), 3.41 (qd,J=5.0, 1.7 Hz, 2H), 2.62-2.49 (m, 4H), 2.19-2.01 (m, 1H), 1.44 (d, J=7.2Hz, 3H), 0.95 (d, J=11.9 Hz, 1H), 0.94 (d, J=11.9 Hz, 1H).

(b) Preparation of LIN 3-2: MC2-PEG4-Val-Ala-PABOH

To a solution of LIN 3-1 (1.84 g, 3.13 mmol) and 4-aminobenzyl alcohol(PABOH) (0.77 g, 6.27 mmol) in CH₂Cl₂ (70 mL) was added a solution ofHOBt (0.84 g, 6.27 mmol) in DMF (5 mL) followed by addition of DIPCDI(0.97 mL, 6.27 mmol) at 23° C. The reaction mixture was stirred for 5 hat 23° C., Et₂O (150 mL) was added, and the solid obtained was filtratedunder vacuum to obtain crude LIN 3-2 (1.74 g, 81% yield) which was usedin the next step without further purification.

¹H NMR (500 MHz, DMSO-d₆): δ 7.58 (d, J=8.5 Hz, 2H), 7.30 (d, J=8.5 Hz,2H), 6.81 (s, 2H), 4.56 (s, 2H), 4.52-4.41 (m, 1H), 4.21 (d, J=6.7 Hz,1H), 3.91 (p, J=6.5 Hz, 1H), 3.81-3.67 (m, 4H), 3.65-3.54 (m, 12H), 3.49(t, J=5.5 Hz, 2H), 2.56 (dd, J=6.6, 5.5 Hz, 2H), 2.46 (t, J=6.9 Hz, 2H),2.12 (h, J=6.8 Hz, 1H), 1.45 (d, J=7.2 Hz, 3H), 1.00 (d, J=12.1 Hz, 3H),0.98 (d, J=12.1 Hz, 3H).

(c) Preparation of LIN 3: MC2-PEG4-Val-Ala-PABC-PNP

To a solution of LIN 3-2 (1.74 g, 2.51 mmol) and bis(4-nitrophenyl)carbonate (bis-PNP) (3.82 g, 12.57 mmol) in CH₂Cl₂:DMF (8:1, 70 mL) wasadded DIPEA (1.31 mL, 7.54 mmol) at 23° C. The reaction mixture wasstirred for 20 h at 23° C. and poured onto silica gel column(CH₂Cl₂:CH₃OH, from 50:1 to 10:1) to afford pure LIN 3 (1.26 g, 59%yield).

¹H NMR (500 MHz, CDCl₃): δ 8.82 (s, 1H), 8.27 (d, J=9.2 Hz, 2H), 7.73(d, J=8.6 Hz, 2H), 7.38 (d, J=9.1 Hz, 4H), 7.15 (dd, J=21.8, 7.2 Hz,2H), 6.69 (s, 2H), 6.62 (t, J=5.7 Hz, 1H), 5.24 (s, 2H), 4.67 (p, J=7.2Hz, 1H), 4.24 (dd, J=6.8, 5.7 Hz, 1H), 3.91-3.76 (m, 2H), 3.71 (ddd,J=10.1, 6.1, 4.3 Hz, 1H), 3.66-3.54 (m, 14H), 3.53 (t, J=5.1 Hz, 1H),3.46-3.33 (m, 2H), 2.76-2.57 (m, 1H), 2.57-2.42 (m, 2H), 2.33-2.19 (m,1H), 1.46 (d, J=7.1 Hz, 3H), 1.01 (d, J=12.1 Hz, 3H), 1.00 (d, J=12.1Hz, 3H).

¹³C NMR (75 MHz, CD3OD): δ 173.0, 172.1, 171.6 (×2), 170.7, 163.8,155.7, 152.5, 145.4, 140.3, 138.9, 134.1, 130.4, 129.1, 125.6, 124.8,121.9, 119.7, 115.1, 70.2, 70.1 (×3), 70.0, 69.9, 69.8, 69.0, 66.9,59.1, 53.4, 49.7, 39.0, 36.0, 34.3, 34.1, 30.4, 18.3, 17.3, 16.6.

ESI-MS m/z: 857.3 (M+H)⁺.

R_(f)=0.45 (CH₂Cl₂:CH₃OH, 9:1).

Example 1: Synthesis of a Compounds of Formula D-X-(AA)_(w)-(T)_(g)-L₁Preparation of Compound DL-1

To a solution of 11-R (100 mg, 0.12 mmol) and LIN 1 (465 mg, 0.63 mmol)in N-Methyl-2-pyrrolidone (NMP) (15 mL) was addedN,N-diisopropylethylamine (DIPEA) (111 μL, 0.63 mmol) at 23° C. Thereaction mixture was stirred for 3 days at 23° C., diluted with EtOAc(50 mL) and washed with H₂O (4×30 mL) and a saturated aqueous solutionof NaCl (30 mL). The combined organic layers were dried over anhydrousNa₂SO₄, filtered, and concentrated under vacuum. The residue waspurified by flash chromatography on silica gel (CH₂Cl₂:CH₃OH, from 99:1to 90:10) to obtain DL 1 which was purified by HPLC preparative to yieldpure DL 1 (69 mg, 40% yield).

¹H NMR (400 MHz, CD3OD/CDCl₃): δ 7.85 (d, J=7.9 Hz, 1H), 7.59 (d, J=8.1Hz, 2H), 7.36 (d, J=8.2 Hz, 2H), 7.30 (d, J=7.9 Hz, 1H), 7.22 (d, J=8.2Hz, 1H), 7.01 (t, J=7.6 Hz, 1H), 6.91 (t, J=7.5 Hz, 1H), 6.72 (s, 2H),6.58 (s, 1H), 6.26 (s, 1H), 6.07 (s, 1H), 5.25-5.14 (m, 2H), 5.14-5.01(m, 2H), 4.67 (bs, 1H), 4.51 (d, J=6.7 Hz, 2H), 4.28 (dd, J=16.2, 7.1Hz, 4H), 4.21-4.05 (m, 3H), 3.71 (s, 3H), 3.51-3.40 (m, 2H), 3.36-3.32(m, 2H), 3.23-2.99 (m, 2H), 2.99-2.72 (m, 2H), 2.65 (d, J=14.9 Hz, 2H),2.28 (s, .3H), 2.25 (s, .3H), 2.10 (s, .3H), 2.04 (s, .3H), 1.96-1.83(m, 1H), 1.80-1.68 (m, 2H), 1.65-1.50 (m, 10H), 1.35-1.23 (m, 2H), 0.95(d, J=6.8 Hz, 3H), 0.94 (d, J=6.8 Hz, 3H).

¹³C NMR (75 MHz, CD3OH/CDCl₃): δ 174.9, 172.5, 171.5, 171.1, 170.7,169.5, 160.8, 157.4, 148.6, 146.1, 143.6, 141.1, 140.9, 138.0, 136.8,133.9, 132.6, 130.5, 129.8, 129.6, 128.6, 126.0, 121.6, 120.4, 119.8,119.0, 118.6, 118.0, 117.8, 116.7, 113.5, 112.8, 110.9, 109.1, 102.3,66.0, 63.1, 62.9, 61.6, 60.2, 59.9, 59.2, 59.1, 58.9, 54.6, 54.6, 53.5,50.7, 45.3, 42.1, 40.5, 37.1, 35.3, 30.3, 29.1, 27.9, 26.3, 26.0, 25.0,24.5, 23.6, 19.4, 18.6, 17.7, 15.1, 8.6.

ESI-MS m/z: 1391.4 (M+H)⁺.

R_(f)=0.40 (CH₂Cl₂:CH₃OH, 9:1).

Preparation of Compound DL 2

To a solution of 11-R (50 mg, 0.063 mmol) and LIN 2 (118 mg, 0.12 mmol)in CH₂Cl₂ (2 mL) was added N,N-diisopropylethylamine (DIPEA) (22 μL,0.12 mmol) at 23° C. The reaction mixture was stirred for 18 h at 23° C.and poured onto silica gel column (CH₂Cl₂:CH₃OH, from 99:1 to 90:10) toyield DL 2 which was purified by HPLC preparative to afford pure DL 2(30 mg, 30% yield).

¹H NMR (400 MHz, CDCl₃): δ 7.63 (d, J=7.9 Hz, 1H), 7.59 (d, J=8.1 Hz,2H), 7.39 (d, J=8.2 Hz, 2H), 7.30 (d, J=7.9 Hz, 1H), 7.23 (d, J=8.2 Hz,1H), 7.01 (t, J=7.6 Hz, 1H), 6.92 (t, J=7.5 Hz, 1H), 6.76 (s, 2H), 6.58(s, 1H), 6.28 (s, 1H), 6.09 (s, 1H), 5.25-5.14 (m, 2H), 5.14-5.01 (m,2H), 4.67 (bs, 1H), 4.51 (d, J=6.7 Hz, 2H), 4.36-4.24 (m, 4H), 4.23-4.15(m, 3H), 3.73 (s, 3H), 3.75-3.69 (m 3H), 3.58-3.50 (m, 14H), 3.51-3.40(m, 2H), 3.36-3.32 (m, 2H), 3.23-3.05 (m, 2H), 2.99-2.88 (m, 2H), 2.68(d, J=14.9 Hz, 2H), 2.56-2.41 (m, 2H), 2.29 (s, .3H), 2.27 (s, .3H),2.10 (s, .3H), 2.05 (s, .3H), 1.96-1.83 (m, 1H), 1.80-1.68 (m, 2H),1.65-1.50 (m, 10H), 1.35-1.23 (m, 2H), 0.96 (d, J=6.8 Hz, 3H), 0.95 (d,J=6.8 Hz, 3H).

¹³C NMR (75 MHz, CD3OD): δ 173.0, 172.34, 171.6, 171.5 (×2), 170.8 (×2),170.7, 169.4, 160.8, 157.4, 155.8, 148.7, 148.5, 146.2, 146.1, 143.7,141.2, 141.1, 140.9, 138.4, 136.9, 134.0, 131.3, 129.8, 128.7, 128.5,126.0, 121.5, 120.5, 119.9, 119.7, 118.5, 117.7, 112.7, 102.4, 70.1(×5), 70.0 (×2), 69.9, 69.8, 69.7, 69.0, 68.9, 66.8, 61.6, 59.9, 59.2,54.6, 54.0, 53.5, 40.4, 40.0, 39.0, 36.0, 35.1, 34.4, 34.1, 30.4, 29.0,26.4, 24.6, 23.6, 19.3, 18.5, 17.4, 15.1, 8.4.

ESI-MS m/z: 1596.6 (M+H)⁺.

R_(f)=0.48 (CH₂Cl₂:CH₃OH, 9:1).

Preparation of Compound DL 3

To a solution of 11-R (50 mg, 0.063 mmol) and LIN 3 (108 mg, 0.12 mmol)in CH₂Cl₂ (2 mL) was added N,N-diisopropylethylamine (DIPEA) (22 μL,0.12 mmol) at 23° C. The reaction mixture was stirred for 18 h at 23° C.and poured onto silica gel column (CH₂Cl₂:CH₃OH, from 99:1 to 90:10) toyield DL 3 which was purified by HPLC preparative to afford pure DL 3(25 mg, 26% yield).

¹H NMR (400 MHz, CD3OD): δ 7.64 (d, J=8.2 Hz, 2H), 7.39 (d, J=8.4 Hz,2H), 7.30 (dt, J=7.8, 1.0 Hz, 1H), 7.23 (dt, J=8.4, 0.9 Hz, 1H), 7.01(ddd, J=8.2, 7.0, 1.2 Hz, 1H), 6.91 (ddd, J=7.9, 7.0, 1.0 Hz, 1H), 6.76(s, 2H), 6.61 (s, 1H), 6.28 (s, 1H), 6.10 (s, 1H), 5.26-5.15 (m, 1H),5.07 (dd, J=12.1, 4.7 Hz, 2H), 4.66 (s, 1H), 4.57 (s, 1H), 4.47 (t,J=7.1 Hz, 1H), 4.36 (s, 1H), 4.32-4.22 (m, 2H), 4.18 (d, J=6.5 Hz, 2H),3.75-3.68 (m, 6H), 3.72 (s, 3H), 3.59-3.46 (m, 8H), 3.44 (t, J=5.3 Hz,2H), 3.33-3.25 (m, 10H), 3.15 (dd, J=9.7, 5.0 Hz, 1H), 2.95 (d, J=17.9Hz, 1H), 2.81 (dd, J=18.0, 9.9 Hz, 2H), 2.73-2.59 (m, 2H), 2.52 (t,J=6.1 Hz, 2H), 2.43 (t, J=6.9 Hz, 2H), 2.29 (s, 3H), 2.27 (s, 3H), 2.11(s, 3H), 2.05 (s, 3H), 1.45 (t, J=8.7 Hz, 2H), 0.99 (d, J=9.9 Hz, 3H),0.97 (d, J=9.9 Hz, 3H).

¹³C NMR (75 MHz, CD3OD): δ 173.1, 172.1, 172.1, 171.6, 171.5, 170.7,169.4, 148.7, 146.1, 143.6, 140.9, 138.1, 136.9, 135.7, 134.0, 134.0,132.7, 127.1, 126.0, 121.5, 120.5, 119.8, 119.7, 119.1, 118.5, 117.6,113.4, 110.8, 105.8, 102.4, 99.9, 86.9, 70.1, 70.1, 70.1, 70.0, 70.0,69.9, 69.8, 69.0, 67.7, 66.8, 65.9, 63.0, 62.0, 61.6, 60.1, 59.9, 59.2,59.1, 54.7, 54.6, 50.8, 49.6, 42.1, 40.3, 40.0, 39.0, 36.0, 34.3, 34.1,30.4, 28.8, 23.6, 19.2, 18.4, 17.3, 16.6, 14.9, 8.4.

ESI-MS m/z: 1511.2 (M+H)⁺.

R_(f)=0.50 (CH₂Cl₂:CH₃OH, 9:1).

Preparation of Compound DL 4

To a solution of 46-R (26 mg, 0.032 mmol) and LIN 1 (48 mg, 0.64 mmol)in DMF (2 mL) was added N,N-diisopropylethylamine (DIPEA) (12 μL, 0.64mmol) at 23° C. The reaction mixture was stirred for 18 hours at 23° C.,diluted with EtOAc (50 mL) and washed with H₂O (4×30 mL) and a saturatedaqueous solution of NaCl (30 mL). The combined organic layers were driedover anhydrous Na₂SO₄, filtered, and concentrated under vacuum. Theresidue was purified by HPLC preparative to yield pure DL 4 (14 mg, 31%yield).

¹H NMR (400 MHz, CD3OD): δ 7.88 (d, J=7.9 Hz, 1H), 7.60 (d, J=8.2 Hz,2H), 7.39-7.36 (m, 3H), 7.20-7.11 (m, 3H), 6.74 (s, 2H), 6.57 (s, 1H),6.24 (s, 1H), 6.08 (s, 1H), 5.30-5.05 (m, 2H), 4.64 (s, 1H), 4.50 (d,J=6.1 Hz, 2H), 4.34-4.27 (m, 4H), 4.21-4.13 (m, 3H), 3.70 (s, 3H), 3.45(t, J=7.2 Hz, 2H), 3.19-3.06 (m, 4H), 2.93-2.76 (m, 2H), 2.70-2.56 (m,2H), 2.29-2.19 (m, 4H), 2.25 (s, 3H), 2.29-2.11 (m, 2H), 2.11 (s, 3H),2.06 (s, 3H), 1.93-1.72 (m, 2H), 1.64-1.52 (m, 9H), 1.32-1.26 (m, 3H),0.96-0.93 (m, 8H).

¹³C NMR (100 MHz, CD3OD): δ 173.4, 171.1, 169.7, 169.3, 169.1, 167.7,159.3, 155.9, 153.5, 147.2, 147.1, 144.3, 142.1, 139.6, 139.2, 136.6,132.5, 131.1, 129.1, 128.3, 127.2, 125.6, 122.7, 121.0, 119.2 (×2),118.3, 117.5, 117.4, 112.2, 112.1, 111.7, 109.7, 100.8, 97.0, 64.6,61.7, 60.0, 58.9, 58.4, 57.7 (×2), 57.4, 53.2, 53.1, 52.0, 48.9, 43.6,40.5, 39.0, 35.6, 33.8, 28.8, 28.1, 27.6, 26.5, 24.6, 23.6, 22.6, 22.2,21.5, 17.9, 17.1, 16.2, 13.6, 7.1.

ESI-MS m/z: 1392.4 (M+H)⁺.

Preparation of Compound DL 5

To a solution of DL 3 (30 mg, 0.026 mmol) in CH₃CN:H₂O (1.39:1, 6 mL,0.015 M) was added AgNO₃ (132 mg, 0.79 mmol). After 18 h at 23° C., thereaction mixture was quenched with an aqueous solution of NaHCO₃ andextracted with CH₂Cl₂ (×3). The combined organic layers were dried overanhydrous Na₂SO₄, filtered, and concentrated under vacuum. The residueobtained was purified in an automatic system for flash chromatography(SiO₂, CH₂Cl₂:CH₃OH, from 95:5 to 50:50) to obtain pure DL 5 (34 mg,87%).

¹H NMR (400 MHz, CD3OD): 7.64 (d, J=8.4 Hz, 2H), 7.38 (d, J=8.3 Hz, 2H),7.29 (d, J=7.8 Hz, 1H), 7.22 (d, J=8.1 Hz, 1H), 7.00 (ddd, J=8.2, 7.0,1.2 Hz, 1H), 6.91 (ddd, J=8.0, 7.0, 1.1 Hz, 1H), 6.75 (s, 2H), 6.64 (s,1H), 6.25 (d, J=1.4 Hz, 1H), 6.06 (d, J=1.4 Hz, 1H), 5.23-5.06 (m, 3H),4.60 (s, 1H), 4.47 (q, J=7.1 Hz, 1H), 4.31 (dd, J=16.3, 4.1 Hz, 2H),4.18 (d, J=6.6 Hz, 1H), 4.12 (d, J=11.6 Hz, 1H), 3.76-3.66 (m, 2H), 3.61(d, J=5.3 Hz, 1H), 3.57-3.48 (m, 16H), 3.44 (t, J=7.2 Hz, 4H), 3.27 (t,J=5.3 Hz, 3H), 3.13 (dd, J=13.4, 6.7 Hz, 1H), 2.96 (d, J=18.0 Hz, 1H),2.85 (dd, J=18.1, 9.3 Hz, 1H), 2.75-2.61 (m, 2H), 2.56-2.48 (m, 2H),2.43 (t, J=6.9 Hz, 2H), 2.28 (s, 6H), 2.24 (s, 1H), 2.14 (s, 3H),2.12-2.05 (m, 2H), 2.04 (s, 3H), 1.43 (d, J=7.2 Hz, 3H), 0.99 (d, J=6.8Hz, 3H), 0.96 (d, J=6.8 Hz, 3H).

¹³C NMR (100 MHz, CD3OD): δ 173.1, 172.1, 171.7, 171.6, 171.5, 170.7,169.3, 162.5, 157.5, 148.9, 146.1, 144.1, 141.4, 140.9, 138.2, 136.9,134.0, 132.7, 131.2, 129.7, 128.4, 126.0, 121.4, 120.1, 119.8, 119.7,118.5, 117.6, 114.3, 112.5, 110.8, 102.3, 90.5, 70.1 (×2), 70.0 (×2),69.9, 69.8, 69.0, 66.8, 65.9, 63.1, 60.2, 59.2, 59.1, 57.9, 55.6, 55.1,53.6, 50.8, 49.6, 45.4, 42.1, 40.3, 39.5, 39.0, 35.9, 34.3, 34.0 (×2),29.3, 24.7, 23.2, 19.1, 18.3, 17.3, 16.5, 15.0, 8.2.

ESI-MS m/z: 1483.4 (M−H₂O+H)⁺.

Preparation of Compound DL 6

To a solution of DL 1 (50 mg, 0.035 mmol) in CH₃CN:H₂O (1.39:1, 2.39 mL,0.015 M) was added AgNO₃ (181 mg, 1.07 mmol). After 18 h at 23° C., thereaction mixture was quenched with an aqueous solution of NaHCO₃:NaCl(1:1) and extracted with CH₂Cl₂ (×3). The combined organic layers weredried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum.The residue obtained was purified by HPLC preparative to obtain pure DL6 (23 mg, 47% yield).

¹H NMR (400 MHz, CD3OD): δ 7.62 (d, J=8.1 Hz, 2H), 7.41 (d, J=8.1 Hz,2H), 7.39 (d, J=8.2 Hz, 1H), 7.29 (d, J=8.2 Hz, 1H), 7.12 (ddd, J=8.0,7.0, 0.9 Hz, 1H), 7.01 (ddd, J=8.0, 7.0, 0.9 Hz, 1H), 6.82 (s, 1H), 6.75(s, 2H), 6.32 (d, J=1.3 Hz, 1H), 6.12 (d, J=1.3 Hz, 1H), 5.32 (d, J=11.8Hz, 1H), 5.24-5.12 (m, 3H), 4.81 (m, 2H), 4.65 (s, 1H), 4.47 (s, 1H),4.31 (d, J=11.6 Hz, 1H), 4.18 (d, J=9.0 Hz, 1H), 4.12 (d, J=7.4 Hz, 1H),3.93-3.83 (m, 2H), 3.76 (s, 3H), 3.65 (d, J=12.3 Hz, 1H), 3.44 (t, J=7.1Hz, 2H), 3.23-2.99 (m, 2H), 2.92 (d, J=15.9 Hz, 1H), 2.62 (s, 3H), 2.45(d, J=15.5 Hz, 1H), 2.35 (s, 3H), 2.30 (s, 3H), 2.28 (d, J=13.3 Hz, 6H),2.07 (s, 3H), 2.13-2.00 (m, 2H), 1.90 (m, 1H), 1.80-1.70 (m, 2H),1.65-1.50 (m, 4H), 1.34-1.22 (m, 2H), 0.96 (d, J=6.8 Hz, 3H), 0.95 (d,J=6.8 Hz, 3H).

¹³C NMR (100 MHz, CD3OD): δ 175.0, 172.6, 171.1, 170.9, 169.2, 169.1,160.9, 157.7, 149.5, 147.0, 146.0, 144.8, 141.7, 141.2, 137.3, 136.8,133.9, 132.5, 130.5, 129.8, 129.6, 128.5, 127.9, 122.6, 120.3, 119.8,119.2, 118.4, 117.9, 116.7, 113.1, 112.8, 111.1, 108.4, 102.7, 101.5,89.1, 66.2, 63.1, 62.9, 61.6, 59.3, 56.9, 56.1, 55.5, 54.7, 53.6, 50.7,45.3, 42.2, 39.0, 38.9, 37.0, 35.1, 30.1, 29.3, 27.9, 26.5, 26.3, 26.0,25.0, 23.1, 19.1, 18.4, 17.6, 15.2, 8.3.

ESI-MS m/z: 1364.4 (M−H₂O+H)⁺.

Preparation of Compound DL 7

To a solution of 12-R (100 mg, 0.12 mmol) and LIN 2 (180 mg, 0.19 mmol)in Dimethylformamide (DMF) (2 mL, 0.06 M) was addedN,N-Diisopropylethylamine (DIPEA) (90 μL, 0.51 mmol) at 23° C. After 18hours the reaction mixture was purified by HPLC preparative to yieldpure DL 7 (125 mg, 62% yield).

¹H NMR (400 MHz, CD3OD): δ 7.65 (d, J=8.1 Hz, 2H), 7.41 (d, J=8.1 Hz,2H), 7.39 (d, J=8.2 Hz, 1H), 7.29 (d, J=8.2 Hz, 1H), 7.13 (ddd, J=8.2,7.0, 1.1 Hz, 1H), 7.01 (ddd, J=8.2, 7.0, 1.1 Hz, 1H), 6.82 (s, 1H), 6.76(s, 2H), 6.32 (d, J=1.3 Hz, 1H), 6.12 (d, J=1.3 Hz, 1H), 5.32 (d, J=11.8Hz, 1H), 5.18 (d, J=5.7 Hz, 2H), 4.82 (m, 2H), 4.64 (d, J=2.8 Hz, 1H),4.47 (s, 1H), 4.33 (dd, J=12.0, 2.1 Hz, 1H), 4.22-4.12 (m, 2H), 3.96(bs, 1H), 3.88 (d, J=5.0 Hz, 1H), 3.78-3.67 (m, 8H), 3.60-3.52 (m, 14H),3.56-3.41 (m, 3H), 3.27 (t, J=5.4 Hz, 2H), 3.22-3.04 (m, 4H), 2.93 (d,J=16.0 Hz, 1H), 2.70-2.58 (m, 3H), 2.55 (t, J=6.9 Hz, 2H), 2.43 (t,J=6.9 Hz, 2H), 2.36 (s, 3H), 2.30 (s, 3H), 2.15-2.07 (m, 1H), 2.06 (s,3H), 2.02 (s, 2H), 1.95-1.87 (m, 1H), 1.80-1.70 (m, 1H), 1.65-1.50 (m,2H), 0.97 (d, J=6.8 Hz, 3H), 0.96 (d, J=6.8 Hz, 3H).

¹³C NMR (125 MHz, CD3OD): δ 173.1, 172.5, 171.6, 171.0, 170.7 (×2),169.3, 168.5, 160.9, 160.0, 149.6, 147.1, 144.9, 141.8, 141.3, 138.2,137.4, 134.1, 132.4, 128.6, 127.9, 125.2, 122.9, 120.2, 119.9, 119.4,118.1 (×2), 117.1, 114.8, 113.2, 112.7, 111.3, 108.3, 102.7, 89.0, 70.2,70.1 (×2), 70.0, 69.9, 69.8, 69.0, 66.9, 66.3, 65.5, 65.4, 61.9, 59.4,56.9, 56.1, 55.4 (×2), 54.6, 53.7, 43.7, 42.4, 39.1, 39.0, 36.0, 34.4,34.1, 31.4, 30.3, 28.9, 26.5, 23.1, 22.3, 19.2, 18.5, 17.5, 15.3, 13.2,8.5.

ESI-MS m/z: 1570.4 (M−H₂O+H)⁺.

Preparation of Compound DL 8

To a solution of 11-S (30 mg, 0.037 mmol) and LIN 1 (56 mg, 0.075 mmol)in Dimethylformamide (DMF) (2 mL, 0.018 M) was addedN,N-Diisopropylethylamine (DIPEA) (26 μL, 0.15 mmol) and1-Hydroxybenzotriazole (HOBt, 10 mg, 0.075 mmol) at 23° C. After 18hours the reaction mixture was purified by HPLC preparative to yieldpure DL 8 (30 mg, 58% yield).

¹H NMR (400 MHz, CD3OD): δ 7.59 (d, J=8.1 Hz, 2H), 7.36 (d, J=8.2 Hz,2H), 7.28 (d, J=7.9 Hz, 1H), 7.23 (d, J=8.2 Hz, 1H), 7.00 (t, J=7.6 Hz,1H), 6.91 (t, J=7.5 Hz, 1H), 6.74 (s, 2H), 6.50 (s, 1H), 6.27 (s, 1H),6.09 (s, 1H), 5.20-5.03 (m, 2H), 4.65 (bs, 1H), 4.54-4.46 (m, 1H),4.43-4.37 (m, 1H), 4.34-4.30 (m, 1H), 4.17-4.12 (m, 1H), 3.75 (s, 3H),3.45 (t, J=7.0 Hz, 4H), 3.32-3.23 (m, 2H), 3.38 (d, J=7.6 Hz, 2H),3.23-2.99 (m, 2H), 3.21-2.97 (m, 3H), 2.94-2.83 (m, 3H), 2.61-2.53 (m,2H), 2.48-2.34 (m, 2H), 2.28 (s, .3H), 2.27-2.22 (m, 1H), 2.21 (s, .3H),2.11 (s, .3H), 2.08-2.02 (m, 1H), 1.99 (s, .3H), 1.91-1.82 (m, 1H),1.77-1.68 (m, 1H), 1.65-1.50 (m, 6H), 1.31-1.24 (m, 2H), 0.94 (d, J=6.8Hz, 3H), 0.93 (d, J=6.8 Hz, 3H).

¹³C NMR (100 MHz, CD3OD): δ 174.9, 174.8, 172.5, 172.0, 171.2, 170.8,169.4, 160.8, 157.5, 148.6, 146.0, 143.6, 140.9, 140.8, 138.0, 136.6,136.5, 134.0, 132.7, 130.2, 129.9, 129.7, 128.7, 128.4, 126.3, 121.6,120.5, 120.0, 119.8, 119.3, 118.7, 118.0, 117.7, 113.5, 112.9, 111.0,107.8, 102.4, 65.9, 63.5, 61.4, 60.6, 59.7, 59.3, 59.2, 59.1, 58.8,54.6, 54.6, 53.4, 44.8, 42.3, 40.6, 38.3, 37.1, 35.3 (×2), 30.3, 29.1,28.0, 26.3, 26.0, 25.1, 24.0, 23.7, 19.5, 18.6, 17.7, 15.2, 8.6.

ESI-MS m/z: 1391.4 (M+H)⁺.

Preparation of Compound DL 9

To a solution of 11-S (110 mg, 0.13 mmol) and LIN-3 (119 mg, 0.13 mmol)in Dimethylformamide (DMF) (4 mL, 0.032 M) was addedN,N-Diisopropylethylamine (DIPEA) (97 μL, 0.55 mmol) and1-Hydroxybenzotriazole (HOBt, 38 mg, 0.27 mmol) at 23° C. After 18 hoursthe reaction mixture was purified by HPLC preparative to yield pure DL 9(120 mg, 57% yield).

¹H NMR (400 MHz, CD3OD): δ 7.62 (d, J=8.6 Hz, 2H), 7.36 (d, J=8.5 Hz,2H), 7.28 (d, J=8.2 Hz, 1H), 7.23 (d, J=8.2 Hz, 1H), 7.00 (ddd, J=8.2,7.0, 1.2 Hz, 1H), 6.91 (td, J=7.5, 7.0, 1.1 Hz, 1H), 6.75 (s, 2H), 6.50(s, 1H), 6.26 (d, J=1.3 Hz, 1H), 6.08 (d, J=1.4 Hz, 1H), 5.19 (d, J=11.4Hz, 1H), 5.09 (d, J=10.4 Hz, 2H), 4.65 (s, 1H), 4.48 (p, J=6.9 Hz, 1H),4.42-4.38 (m, 2H), 4.34-4.30 (m, 2H), 4.22-4.14 (m, 1H), 3.78-3.64 (m,5H), 3.61-3.50 (m, 8H), 3.45 (t, J=5.4 Hz, 2H), 3.38 (d, J=5.1 Hz, 1H),3.33-3.23 (m, 3H), 3.02 (dd, J=13.5, 5.5 Hz, 1H), 2.89 (d, J=9.4 Hz,2H), 2.62-2.33 (m, 7H), 2.27 (s, 3H), 2.22 (s, 3H), 2.12 (s, 3H),2.15-2.05 (m, 1H), 1.99 (m, 3H), 1.42 (d, J=7.1 Hz, 3H), 0.97 (dd, J=6.8Hz, 3H), 0.95 (dd, J=6.8 Hz, 3H).

¹³C NMR (100 MHz, CD3OD): δ 173.1 (×2), 172.1, 171.9, 171.6, 171.5,171.5, 170.7, 169.3, 148.6, 146.0, 143.6, 140.9, 140.8, 138.1, 136.5,134.1, 132.8, 130.3, 129.8, 128.4, 126.3, 121.6, 120.5, 120.0, 119.7,119.3, 118.7, 118.0, 117.7, 113.5, 112.9, 111.0, 107.8, 102.4, 70.2,70.1 (×2), 70.0 (×2), 69.8, 69.1, 66.9, 65.9, 63.5, 61.4, 60.6, 59.7,59.3, 59.2, 59.1, 58.8, 54.7, 54.6, 49.6, 42.3, 40.5, 39.1, 39.0, 36.0,34.4 (×2), 34.1, 30.4, 24.0, 23.7, 19.4, 18.5, 17.4, 16.8, 15.1, 8.5.

ESI-MS m/z: 1511.4 (M+H)⁺.

Preparation of Compound DL 10

To a solution of 12-S (30 mg, 0.058 mmol) and LIN 1 (98 mg, 0.13 mmol)in N-Methyl-2-pyrrolidone (NMP) (4 mL, 0.014 M) was addedN,N-Diisopropylethylamine (DIPEA) (83 μL, 0.13 mmol) at 23° C. After 18hours the reaction mixture was diluted with EtOAc (25 mL) and washedwith H₂O (4×25 mL) and an aqueous saturated solution of NaCl. Thecombined organic layers were dried over anhydrous Na₂SO₄, filtered, andconcentrated under vacuum. The residue was purified by flashchromatography on silica gel (CH₂Cl₂:CH₃OH, from 99:1 to 90:10) and thecompound obtained purified by HPLC preparative to yield pure DL 10 (11mg, 21% yield).

¹H NMR (400 MHz, CD3OD): δ 7.61 (d, J=8.1 Hz, 2H), 7.39 (d, J=8.1 Hz,2H), 7.35 (d, J=8.2 Hz, 1H), 7.29 (d, J=8.2 Hz, 1H), 7.08 (ddd, J=8.0,7.0, 0.9 Hz, 1H), 6.98 (ddd, J=8.0, 7.0, 0.9 Hz, 1H), 6.76 (s, 2H), 6.66(s, 1H), 6.29 (d, J=1.3 Hz, 1H), 6.14 (d, J=1.3 Hz, 1H), 5.13 (q, J=12.3Hz, 2H), 4.93-.4.81 (m, 3H), 4.69 (s, 1H), 4.48 (s, 1H), 4.23 (d, J=10.0Hz, 1H), 4.13 (dd, J=7.5, 4.0 Hz, 1H), 3.90 (d, J=5.0, 1H), 3.76 (s,3H), 3.49-3.41 (m, 2H), 3.40-3.27 (m, 1H), 3.28-3.24 (m, 4H), 3.22-3.05(m, 4H), 2.80-2.65 (m, 3H), 2.63 (s, 3H), 2.29 (s, 3H), 2.28 (s, 3H),2.13-2.00 (m, 2H), 2.03 (s, 3H), 1.93-1.83 (m, 1H), 1.79-1.69 (m, 2H),1.65-1.51 (m, 6H), 1.34-1.22 (m, 2H), 0.96 (d, J=6.8 Hz, 3H), 0.95 (d,J=6.8 Hz, 3H).

¹³C NMR (100 MHz, CD3OD): δ 174.9, 172.7, 172.6, 171.1, 170.8 (×2),169.1, 160.9, 149.4, 146.6, 144.7, 142.3, 141.5, 141.0, 138.1, 136.8,133.9, 133.5, 132.6, 128.5, 127.5, 125.9, 122.2, 120.9, 119.8, 117.8,113.2, 113.0, 111.2, 107.3, 102.6, 89.2, 66.2, 61.7, 61.3, 60.2, 59.4,59.3, 57.4, 56.9, 55.8, 55.7, 55.2, 54.8, 53.5, 42.0, 39.1, 37.1, 35.2,31.7, 30.2, 29.4, 29.1, 29.0 (×2), 27.9, 26.4, 26.0, 25.0, 23.3, 22.4,19.2, 18.5, 17.7, 15.2, 13.2, 8.5.

ESI-MS m/z: 1364.4 (M−H₂O+H)⁺.

Preparation of Compound DL 11

To a solution of DL 9 (90 mg, 0.059 mmol) in CH₃CN:H₂O (1.39:1, 4 mL,0.015 M) was added AgNO₃ (298 mg, 1.78 mmol). After 21 h at 23° C., thereaction mixture was quenched with an aqueous solution of NaHCO₃:NaCl(1:1) and extracted with CH₂Cl₂ (×3). The combined organic layers weredried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum.The residue obtained was purified in an automatic system for flashchromatography (SiO₂, CH₂Cl₂:CH₃OH, from 95:5 to 50:50) to obtain pureDL 11 (65 mg, 73% yield).

¹H NMR (400 MHz, CD3OD): δ 7.62 (d, J=8.6 Hz, 1H), 7.37 (d, J=8.5 Hz,2H), 7.28 (d, J=7.8 Hz, 1H), 7.23 (d, J=8.2 Hz, 1H), 6.99 (ddd, J=8.2,7.0, 1.2 Hz, 1H), 6.90 (ddd, J=8.0, 7.0, 1.1 Hz, 1H), 6.76 (s, 2H), 6.52(s, 1H), 6.25 (d, J=1.3 Hz, 1H), 6.06 (d, J=1.3 Hz, 1H), 5.28 (d, J=11.4Hz, 1H), 5.09 (d, J=10.4 Hz, 1H), 4.59 (s, 1H), 4.47 (q, J=7.1 Hz, 1H),4.33 (dd, J=11.5, 1.9 Hz, 1H), 4.33-4.25 (m, 2H), 4.19 (d, J=6.6 Hz,1H), 3.75 (s, 3H), 3.75-3.67 m, 3H), 3.61 (d, J=5.3 Hz, 1H), 3.58-3.51(m, 12H), 3.45 (dd, J=10.6, 5.2 Hz, 2H), 3.34 (s, 3H), 3.27 (t, J=5.5Hz, 3H), 3.04 (dd, J=13.4, 6.7 Hz, 1H), 2.88-2.82 (m, 3H), 2.64-2.35 (m,7H), 2.27 (s, 3H), 2.23 (s, 3H), 2.17-2.04 (m, 1H), 2.09 (s, 3H), 2.01(d, J=7.2 Hz, 1H), 1.98 (s, 3H), 1.42 (d, J=7.2 Hz, 3H), 0.98 (dd, J=6.8Hz, 3H), 0.95 (dd, J=6.8 Hz, 3H).

¹³C NMR (100 MHz, CD3OD): δ 173.1, 172.1, 172.0, 171.6, 171.5, 170.7,169.4, 157.6, 148.6, 145.8, 143.7, 141.1, 140.8, 138.1, 136.5, 134.1,132.8, 130.4, 130.0, 129.8, 128.4, 126.4, 126.2, 121.5, 120.9, 120.1,119.8, 118.6, 117.7, 114.9, 112.4, 111.0, 107.7, 102.1, 91.0, 70.2, 70.1(×2), 70.0 (×2), 69.8, 69.1, 66.9, 65.9, 63.5, 60.5, 59.3, 59.1, 58.2,55.2, 54.7, 52.9, 49.6, 44.9, 42.6, 40.0, 39.0, 38.3, 36.0, 34.4, 34.1,30.4, 29.4, 24.0, 23.3, 19.4, 18.5, 17.4, 16.8, 15.1, 8.4.

ESI-MS m/z: 1483.4 (M−H₂O+H)⁺.

Example 2: Preparation of Antibody-Drug Conjugates (ADCs)

In this Example, syntheses of antibody-drug conjugates of the presentinvention are described. It should be noted that these syntheses areexemplary and that the processes described can be applied to all thecompounds and antibodies described herein.

Example 2a Preparation of Anti-CD13 Monoclonal Antibody

Anti-CD13 monoclonal antibodies were obtained following well knownprocedures commonly used in the art. Briefly BALB/c mice were immunizedwith human endothelial cells isolated from umbilical cord. To that end,1.5E7 of the cells were injected to the mice intraperitoneally on days−45 and −30 and intravenously on day −3. On day 0 spleen from theseanimals were removed and spleen cells were fused with SP2 mouse myelomacells at a ratio of 4:1 according to standard techniques to produce thehybridoma and distributed on 96-well tissue culture plates (CostarCorp., Cambridge, Mass.). After 2 weeks hybridoma culture supernatantswere harvested and their reactivity against the cell line used in theimmunization step was tested by flow cytometry. Positive supernatantswere assayed by immunofluorescence staining the corresponding cells usedas antigens. Hybridomas showing a specific staining, immunoprecipitationpattern and cell distribution were selected and cloned and subcloned bylimiting dilution.

Once the clones were selected, cells were cultured in RPMI-1640 mediumsupplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 100 U/mLpenicillin and 100 μg/mL streptomycin at 37° C. during 3-4 days untilthe medium turned pale yellow. At that point, two thirds of the mediumvolume were removed, centrifuged at 1,000×g for 10 min to pellet thecells and the supernatant was either centrifuged again for furthercleaning at 3,000×g for 10 min or filtered through 22 μm pore sizemembranes. The clarified supernatant was subjected to precipitation with55% saturation ammonium sulphate and the resulting pellet wasresuspended in 100 mM Tris-HCl pH 7.8 (1 mL per 100 mL of the originalclarified supernatant) and dialyzed at 4° C. for 16-24 h against 5 L of100 mM Tris-HCl pH 7.8 with 150 mM NaCl, changing the dialyzing solutionat least three times. The dialyzed material was finally loaded onto aProtein A-Sepharose column and the corresponding monoclonal antibody waseluted with 100 mM sodium citrate pH 3.0 or alternatively with 1Mglycine pH 3.0. Those fractions containing the antibody were neutralizedwith 2M Tris-HCl pH 9.0 and finally dialyzed against PBS and stored at−80° C. until its use.

Preparation of Antibody-Drug Conjugate ADC1 with Trastuzumab and DL 1(a) Partial Reduction of Trastuzumab to Give Partially ReducedTrastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (13.9 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (0.33 mL, 4.6 mg, 30.6 nmol) was diluted to aconcentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (17.5 μL, 87.6 nmol, 3 eq.) The reduction reaction was left tostir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of4.0.

(b) Preparation of ADC 1

To the solution of partially reduced Trastuzumab (140 μL, 1.4 mg, 9nmol), N,N-Dimethylacetamide (DMA) was added (28.2 μL) followed byaddition of a freshly prepared solution of DL 1 (10 mM in DMA, 6.8 μL,67.5 nmol, 7.5 eq.) Upon addition of DL 1, the solution turned turbid.The conjugation reaction was stirred for 30 min at 20° C. and theturbidity vanished during the conjugation reaction. The excess of drugwas quenched by addition of N-acetylcysteine (NAC) (10 mM, 6.8 μL, 67.5nmol) followed by stirring the solution for 20 min. The quenchedconjugation reaction was purified by desalting using Sephadex G25 NAP-5columns into PBS buffer. The final target product ADC 1 was concentratedto a final concentration of 6.05 mg/mL as determined by UV and 232 μL(1.4 mg, 9.3 nmol, 103%) ADC solution was obtained. HIC HPLC runs wereperformed to determine the percentage of conjugation reaction (89%).

Preparation of Antibody-Drug Conjugate ADC 2 with Trastuzumab and DL 2(a) Partial Reduction of Trastuzumab to Give Partially ReducedTrastuzumab

Trastuzumab solution (0.33 mL, 4.6 mg, 30.6 nmol) was diluted to aconcentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (17.5 μL, 87.6 nmol, 3 eq.) The reduction reaction was left tostir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of3.9.

(b) Preparation of ADC 2

To the solution of partially reduced Trastuzumab (140 μL, 1.4 mg, 9nmol), N,N-Dimethylacetamide (DMA) was added (28.2 μL) followed byaddition of a freshly prepared solution of DL 2 (10 mM in DMA, 6.8 μL,67.5 nmol, 7.5 eq.) Upon addition of DL 2 the solution was stirred for30 min at 20° C. The excess of drug was quenched by addition ofN-acetylcysteine (NAC) (10 mM, 6.8 μL, 67.5 nmol) followed by stirringthe solution for 20 min. The quenched conjugation reaction was purifiedby desalting using Sephadex G25 NAP-5 columns into PBS buffer. The finaltarget product ADC 2 was concentrated to a final concentration of 5.19mg/mL as determined by UV and 270 μL (1.4 mg, 9.3 nmol, 103%) ADCsolution was obtained. HIC HPLC runs were performed to determine thepercentage of conjugation reaction (65%).

Preparation of Antibody-Drug Conjugate ADC 3 with Trastuzumab andCompound DL 3 (a) Partial Reduction of Trastuzumab to Give PartiallyReduced Trastuzumab

Trastuzumab solution (0.33 mL, 4.6 mg, 30.6 nmol) was diluted to aconcentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (17.5 μL, 87.6 nmol, 3 eq.) The reduction reaction was left tostir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of3.9.

(b) Preparation of ADC 3

To the solution of partially reduced Trastuzumab (140 μL, 1.4 mg, 9nmol), N,N-Dimethylacetamide (DMA) was added (28.2 μL) followed byaddition of a freshly prepared solution of DL 3 (10 mM in DMA, 6.8 μL,67.5 nmol, 7.5 eq.) Upon addition of DL 3 the solution was stirred for30 min at 20° C. The excess of drug was quenched by addition ofN-acetylcysteine (NAC) (10 mM, 6.8 μL, 67.5 nmol) followed by stirringthe solution for 20 min. The quenched conjugation reaction was purifiedby desalting using Sephadex G25 NAP-5 columns into PBS buffer. The finaltarget product ADC 3 was concentrated to a final concentration of 5.15mg/mL as determined by UV and 280 μL (1.44 mg, 9.6 nmol, 107%) ADCsolution was obtained. HIC HPLC runs were performed to determine thepercentage of conjugation reaction (93%).

Preparation of Antibody-Drug Conjugate ADC 4 with Traut's-ModifiedTrastuzumab and Compound DL 1 (a) Reaction of Trastuzumab with2-iminothiolane hydrochloride (Traut's reagent) to give thiol-activatedTrastuzumab

Trastuzumab solution (0.65 mL, 9 mg, 60 nmol) was diluted to aconcentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mMEDTA, pH 8). Traut's reagent was added (64.4 μL, 900 nmol, 15 eq.) andthe reaction stirred for 2 h at 25° C. The mixture was buffer exchangedusing two Sephadex G25 NAP-5 columns into PBS buffer, and concentratedto a volume of 1.2 mL (7.5 mg/mL). Immediately after, an Ellman assaywas performed to give a Free Thiol to Antibody ratio (FTAR) of 7.9.

(b) Preparation of ADC 4

To the solution of thiol-activated Trastuzumab (300 μL, 2.25 mg, 15nmol), DMA was added (59.8 μL) followed by addition of a freshlyprepared solution of DL 1 (10 mM in DMA, 22.5 μL, 225 nmol, 15 eq.).Upon addition of DL 1, the solution turned turbid. The conjugationreaction was stirred for 2 h at 25° C. and purified by desalting using aSephadex G25 NAP-5 column into PBS buffer. The final target product ADC4 was concentrated to a final concentration of 3.49 mg/mL as determinedby UV and 252 μL (0.88 mg, 5.86 nmol, 39%) ADC solution was obtained.

Preparation of Antibody-Drug Conjugate ADC 5 with Trastuzumab andCompound DL 4 (a) Partial Reduction of Trastuzumab to Give PartiallyReduced Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (17.1 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (0.5 mL, 8.55 mg, 57 nmol) was diluted to aconcentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (24.5 μL, 122.4 nmol, 2.2 eq.) The reduction reaction was leftto stir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of3.4.

(b) Preparation of ADC 5

To the solution of partially reduced Trastuzumab (200 μL, 1.9 mg, 13.2nmol), N,N-Dimethylacetamide (DMA) was added (42.1 μL) followed byaddition of a freshly prepared solution of DL 4 (10 mM in DMA, 7.9 μL,79.2 nmol, 6 eq.) Upon addition of DL 4, the solution turned turbid. Theconjugation reaction was stirred for 30 min at 20° C. and the turbidityvanished during the conjugation reaction. The excess of drug wasquenched by addition of N-acetylcysteine (NAC) (10 mM, 7.9 μL, 79.2nmol) followed by stirring the solution for 20 min. The quenchedconjugation reaction was purified by desalting using Sephadex G25 NAP-5columns into PBS buffer. The final target product ADC 5 was concentratedto a final concentration of 5.30 mg/mL as determined by UV and 290 μL(1.54 mg, 1.0 nmol, 81%) ADC solution was obtained. HIC HPLC runs wereperformed to determine the percentage of conjugation reaction (91%).

Preparation of Antibody-Drug Conjugate ADC 6 with Trastuzumab andCompound DL 5 (a) Partial Reduction of Trastuzumab to Give PartiallyReduced Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (17.6 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (0.55 mL, 9.7 mg, 64.6 nmol) was diluted to aconcentration of 12.8 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (29.2 μL, 146 nmol, 2.2 eq.) The reduction reaction was left tostir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of3.4.

(b) Preparation of ADC 6

To the solution of partially reduced Trastuzumab (140 μL, 1.8 mg, 12nmol), N,N-Dimethylacetamide (DMA) was added (28.2 μL) followed byaddition of a freshly prepared solution of DL 5 (10 mM in DMA, 6.8 μL,67.5 nmol, 5.6 eq.) Upon addition of DL 5 the solution was stirred for30 min at 20° C. The excess of drug was quenched by addition ofN-acetylcysteine (NAC) (10 mM, 6.8 μL, 67.5 nmol) followed by stirringthe solution for 20 min. The quenched conjugation reaction was purifiedby desalting using Sephadex G25 NAP-5 columns into PBS buffer. The finaltarget product ADC 6 was concentrated to a final concentration of 4.29mg/mL as determined by UV and 320 μL (1.37 mg, 9.1 nmol, 76%) ADCsolution was obtained. HIC HPLC runs were performed to determine thepercentage of conjugation reaction (83%).

Preparation of Antibody-Drug Conjugate ADC 7 with Anti-CD13 and CompoundDL 1 (a) Partial Reduction of Anti-CD13 to Give Partially ReducedAnti-CD13

Anti-CD13 solution (0.5 mL, 8.2 mg, 54.6 nmol) was diluted to aconcentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (31.9 μL, 159 nmol, 3 eq.) The reduction reaction was left tostir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of4.7.

(b) Preparation of ADC 7

To the solution of partially reduced Anti-CD13 (200 μL, 2.0 mg, 13.3nmol), N,N-Dimethylacetamide (DMA) was added (40 μL) followed byaddition of a freshly prepared solution of DL 1 (10 mM in DMA, 10 μL,100 nmol, 7.5 eq.) Upon addition of DL 1 the solution was stirred for 30min at 20° C. The excess of drug was quenched by addition ofN-acetylcysteine (NAC) (10 mM, 10 μL, 100 nmol) followed by stirring thesolution for 20 min. The quenched conjugation reaction was purified bydesalting using Sephadex G25 NAP-5 columns into PBS buffer. The finaltarget product ADC 7 was concentrated to a final concentration of 5.58mg/mL as determined by UV and 350 μL (1.95 mg, 13 nmol, 98%) ADCsolution was obtained. HIC HPLC runs were performed to determine thepercentage of conjugation reaction (90%).

Preparation of Antibody-Drug Conjugate ADC 8 with Anti-CD13 and CompoundDL 3 (a) Partial Reduction of Anti-CD13 to Give Partially ReducedAnti-CD13

Anti-CD13 solution (0.5 mL, 8.2 mg, 54.6 nmol) was diluted to aconcentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (31.9 μL, 159 nmol, 3 eq.) The reduction reaction was left tostir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of4.7.

(b) Preparation of ADC 8

To the solution of partially reduced Anti-CD13 (200 μL, 2.0 mg, 13.3nmol), N,N-Dimethylacetamide (DMA) was added (40 μL) followed byaddition of a freshly prepared solution of DL 3 (10 mM in DMA, 10 μL,100 nmol, 7.5 eq.) Upon addition of DL 3 the solution was stirred for 30min at 20° C. The excess of drug was quenched by addition ofN-acetylcysteine (NAC) (10 mM, 10 μL, 100 nmol) followed by stirring thesolution for 20 min. The quenched conjugation reaction was purified bydesalting using Sephadex G25 NAP-5 columns into PBS buffer. The finaltarget product ADC 8 was concentrated to a final concentration of 5.83mg/mL as determined by UV and 380 μL (2.21 mg, 14.7 nmol, 111%) ADCsolution was obtained. HIC HPLC runs were performed to determine thepercentage of conjugation reaction (94%).

Preparation of Antibody-Drug Conjugate ADC 9 with Anti-CD13 and CompoundDL 5 (a) Partial Reduction of Anti-CD13 to Give Partially ReducedAnti-CD13

Anti-CD13 solution (0.5 mL, 8.2 mg, 54.6 nmol) was diluted to aconcentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (31.9 μL, 159 nmol, 3 eq.) The reduction reaction was left tostir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of4.7.

(b) Preparation of ADC 9

To the solution of partially reduced Anti-CD13 (200 μL, 2.0 mg, 13.3nmol), N,N-Dimethylacetamide (DMA) was added (40 μL) followed byaddition of a freshly prepared solution of DL 5 (10 mM in DMA, 10 μL,100 nmol, 7.5 eq.) Upon addition of DL 5 the solution was stirred for 30min at 20° C. The excess of drug was quenched by addition ofN-acetylcysteine (NAC) (10 mM, 10 μL, 100 nmol) followed by stirring thesolution for 20 min. The quenched conjugation reaction was purified bydesalting using Sephadex G25 NAP-5 columns into PBS buffer. The finaltarget product ADC 9 was concentrated to a final concentration of 5.82mg/mL as determined by UV and 380 μL (2.21 mg, 14.7 nmol, 111%) ADCsolution was obtained. HIC HPLC runs were performed to determine thepercentage of conjugation reaction (89%).

Preparation of Antibody-Drug Conjugate ADC 10 with Anti-CD13 andCompound DL 2 (a) Partial Reduction of Anti-CD13 to Give PartiallyReduced Anti-CD13

Anti-CD13 solution (0.5 mL, 8.2 mg, 54.6 nmol) was diluted to aconcentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (31.9 μL, 159.6 nmol, 3 eq.) The reduction reaction was left tostir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of4.7.

(b) Preparation of ADC 10

To the solution of partially reduced Anti-CD13 (200 μL, 2 mg, 13.3nmol), DMA was added (40 μL) followed by addition of a freshly preparedsolution of DL 2 (10 mM in DMA, 10 μL, 100 nmol, 7.5 eq.). Upon additionof DL 2, the solution turned turbid. The conjugation reaction wasstirred for 30 min at 20° C. The excess of drug was quenched by additionof N-acetylcysteine (NAC) (10 mM, 10 μL, 100 nmol) followed by stirringthe solution for 20 min. The quenched conjugation reaction was purifiedby desalting using Sephadex G25 NAP-5 columns into PBS buffer. The finaltarget product ADC 10 was concentrated to a final concentration of 6.61mg/mL as determined by UV and 250 μL (1.65 mg, 11 nmol, 85%) ADCsolution was obtained. HIC HPLC runs were performed to determine thepercentage of conjugation reaction (23%).

Preparation of Antibody-Drug Conjugate ADC 11 with Trastuzumab andCompound DL 6 (a) Partial Reduction of Trastuzumab to Give PartiallyReduced Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (13.9 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (0.33 mL, 4.6 mg, 30.6 nmol) was diluted to aconcentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (17.5 μL, 87.6 nmol, 3 eq.) The reduction reaction was left tostir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of4.0.

(b) Preparation of ADC 11

To the solution of partially reduced Trastuzumab (140 μL, 1.4 mg, 9nmol), DMA was added (28.2 μL) followed by addition of a freshlyprepared solution of DL 6 (10 mM in DMA, 6.8 μL, 67.5 nmol, 7.5 eq.).Upon addition of DL 6, the solution turned turbid. The conjugationreaction was stirred for 30 min at 20° C. The excess of drug wasquenched by addition of N-acetylcysteine (NAC) (10 mM, 6.8 μL, 67.5nmol) followed by stirring the solution for 20 min. The quenchedconjugation reaction was purified by desalting using Sephadex G25 NAP-5columns into PBS buffer. The final target product ADC 11 wasconcentrated to a final concentration of 6.14 mg/mL as determined by UVand 218 μL (1.33 mg, 8.9 nmol, 99%) ADC solution was obtained. HIC HPLCruns were performed to determine the percentage of conjugation reaction(38%).

Preparation of Antibody-Drug Conjugate ADC 12 with Trastuzumab andCompound DL 7 (a) Partial Reduction of Trastuzumab to Give PartiallyReduced Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (17.1 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (0.5 mL, 8.5 mg, 57 nmol) was diluted to aconcentration of 10 mg/mL with phosphate buffer (50 mM, pH 8). Partialreduction of the disulfide bonds in the antibody was performed by theaddition of a 5.0 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP)solution (24.5 μL, 122.4 nmol, 2.2 eq.) The reduction reaction was leftto stir for 90 min at 20° C. Immediately after the reduction, an Ellmanassay was performed to give a Free Thiol to Antibody ratio (FTAR) of3.4.

(b) Preparation of ADC 12

To the solution of partially reduced Trastuzumab (200 μL, 2 mg, 13.2nmol), DMA was added (42.1 μL) followed by addition of a freshlyprepared solution of DL 7 (10 mM in DMA, 7.9 μL, 79 nmol, 6 eq.). Uponaddition of DL 7, the solution turned turbid. The conjugation reactionwas stirred for 30 min at 20° C. The excess of drug was quenched byaddition of N-acetylcysteine (NAC) (10 mM, 7.9 μL, 79 nmol) followed bystirring the solution for 20 min. The quenched conjugation reaction waspurified by desalting using Sephadex G25 NAP-5 columns into PBS buffer.The final target product ADC 12 was concentrated to a finalconcentration of 5.38 mg/mL as determined by UV and 270 μL (1.45 mg, 9.6nmol, 72%) ADC solution was obtained. HIC HPLC runs were performed todetermine the percentage of conjugation reaction (76%).

Preparation of Antibody-Drug Conjugate ADC 13 with Traut's ModifiedTrastuzumab and Compound DL 2 (a) Reaction of Trastuzumab with2-iminothiolane (Traut's Reagent) to give thiol-activated Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (16.1 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (0.5 mL, 8.0 mg, 53.7 nmol) was diluted to aconcentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mMEDTA, pH 8). Traut's reagent was added (14 mM, 46.0 μL, 644 nmol, 12eq.), and the reaction stirred for 2 h at 20° C. The mixture was bufferexchanged using Sephadex G25 NAP-5 columns into PBS buffer, andconcentrated to a volume of 0.8 mL. Immediately after, an Ellman assaywas performed to give a Free Thiol to Antibody ratio (FTAR) of 4.4.

(b) Preparation of ADC 13

To the solution of thiol-activated Trastuzumab (200 μL, 2.0 mg, 13nmol), DMA was added (37 μL) followed by addition of a freshly preparedsolution of DL 2 (10 mM in DMA, 13 μL, 130 nmol, 10 eq.). Upon additionof DL 2, the solution turned turbid. The conjugation reaction wasstirred for 2 h at 25° C. and purified by desalting using a Sephadex G25NAP-5 column into PBS buffer. The final target product ADC 13 wasconcentrated to a final concentration of 2.83 mg/mL as determined by UVand 340 μL (0.96 mg, 6.4 nmol, 49%) ADC solution was obtained.

Preparation of Antibody-Drug Conjugate ADC 14 with Traut's modifiedTrastuzumab and Compound DL 3 (a) Reaction of Trastuzumab with2-iminothiolane (Trauct's Reagent) to give thiol-activated Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (16.1 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (0.5 mL, 8.0 mg, 53.7 nmol) was diluted to aconcentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mMEDTA, pH 8). Traut's reagent was added (14 mM, 46.0 μL, 644 nmol, 12eq.), and the reaction stirred for 2 h at 20° C. The mixture was bufferexchanged using Sephadex G25 NAP-5 columns into PBS buffer, andconcentrated to a volume of 0.8 mL. Immediately after, an Ellman assaywas performed to give a Free Thiol to Antibody ratio (FTAR) of 4.4.

(b) Preparation of ADC 14

To the solution of thiol-activated Trastuzumab (200 μL, 2.0 mg, 13nmol), DMA was added (37 μL) followed by addition of a freshly preparedsolution of DL 3 (10 mM in DMA, 13 μL, 130 nmol, 10 eq.). Upon additionof DL 3, the solution turned turbid. The conjugation reaction wasstirred for 2 h at 25° C. and purified by desalting using a Sephadex G25NAP-5 column into PBS buffer. The final target product ADC 14 wasconcentrated to a final concentration of 0.75 mg/mL as determined by UVand 380 μL (0.28 mg, 1.9 nmol, 15%) ADC solution was obtained.

Preparation of Antibody-Drug Conjugate ADC 15 with Traut's modifiedTrastuzumab and Compound DL 5 (a) Reaction of Trastuzumab with2-Iminothiolane (Traut's Reagent) to Give Thiol-Activated Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (16.1 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (0.5 mL, 8.0 mg, 53.7 nmol) was diluted to aconcentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mMEDTA, pH 8). Traut's reagent was added (14 mM, 46.0 μL, 644 nmol, 12eq.), and the reaction stirred for 2 h at 20° C. The mixture was bufferexchanged using Sephadex G25 NAP-5 columns into PBS buffer, andconcentrated to a volume of 0.8 mL. Immediately after, an Ellman assaywas performed to give a Free Thiol to Antibody ratio (FTAR) of 4.4.

(b) Preparation of ADC 15

To the solution of thiol-activated Trastuzumab (200 μL, 2.0 mg, 13nmol), DMA was added (37 μL) followed by addition of a freshly preparedsolution of DL 5 (10 mM in DMA, 13 μL, 130 nmol, 10 eq.). Upon additionof DL 5, the solution turned turbid. The conjugation reaction wasstirred for 2 h at 25° C. and purified by desalting using a Sephadex G25NAP-5 column into PBS buffer. The final target product ADC 15 wasconcentrated to a final concentration of 1.79 mg/mL as determined by UVand 440 μL (0.79 mg, 5.2 nmol, 40%) ADC solution was obtained.

Preparation of Antibody-Drug Conjugate ADC 16 with Traut's modifiedTrastuzumab and Compound DL 6 (a) Reaction of Trastuzumab with2-iminothiolane hydrochloride (Traut's reagent) to give thiol-activatedTrastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (17.1 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (0.25 mL, 4.3 mg, 28.5 nmol) was diluted to aconcentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mMEDTA, pH 8). Traut's reagent was added (14 mM, 24.4 μL, 342 nmol, 12eq.), and the reaction stirred for 2 h at 20° C. The mixture was bufferexchanged using Sephadex G25 NAP-5 columns into PBS buffer, andconcentrated to a volume of 0.43 mL. Immediately after, an Ellman assaywas performed to give a Free Thiol to Antibody ratio (FTAR) of 4.6.

(b) Preparation of ADC 16

To the solution of thiol-activated Trastuzumab (200 μL, 2.0 mg, 13nmol), DMA was added (37 μL) followed by addition of a freshly preparedsolution of DL 6 (10 mM in DMA, 13 μL, 130 nmol, 10 eq.). Upon additionof DL 6, the solution turned turbid. The conjugation reaction wasstirred for 2 h at 25° C. and purified by desalting using a Sephadex G25NAP-5 column into PBS buffer. The final target product ADC 16 wasconcentrated to a final concentration of 5.63 mg/mL as determined by UVand 230 μL (1.29 mg, 8.6 nmol, 66%) ADC solution was obtained.

Preparation of Antibody-Drug Conjugate ADC 17 with Traut's modifiedTrastuzumab and Compound DL 8 (a) Reaction of Trastuzumab with2-iminothiolane (Traut's reagent) to give thiol-activated Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (17.7 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (1.5 mL, 26.5 mg, 177 nmol) was diluted to aconcentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mMEDTA, pH 8) and fractionated in two vials (1.3 mL each). Traut's reagentwas added (14 mM, 61.8 μL, 866 nmol, 10 eq.) in each vial, and thereactions stirred for 2 h at 20° C. The reactions were mixed and bufferexchanged using Sephadex G25 NAP-10 columns into PBS buffer, andconcentrated to a volume of 2.6 mL. Immediately after, an Ellman assaywas performed to give a Free Thiol to Antibody ratio (FTAR) of 5.6.

(b) Preparation of ADC 17

To the solution of thiol-activated Trastuzumab (500 μL, 5.0 mg, 33nmol), DMA was added (98.6 μL) followed by addition of a freshlyprepared solution of DL 8 (10 mM in DMA, 26.4 μL, 264 nmol, 8 eq.). Uponaddition of DL 8, the solution turned turbid. The conjugation reactionwas stirred for 2 h at 25° C. and purified by desalting using a SephadexG25 NAP-5 column into PBS buffer. The final target product ADC 17 wasconcentrated to a final concentration of 3.21 mg/mL as determined by UVand 390 μL (1.25 mg, 8.3 nmol, 25%) ADC solution was obtained.

Preparation of Antibody-Drug Conjugate ADC 18 with Traut's modifiedTrastuzumab and Compound DL 9 (a) Reaction of Trastuzumab with2-iminothiolane (Traut's reagent) to give thiol-activated Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (17.7 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (1.5 mL, 26.5 mg, 177 nmol) was diluted to aconcentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mMEDTA, pH 8) and fractionated in two vials (1.3 mL each). Traut's reagentwas added (14 mM, 61.8 μL, 866 nmol, 10 eq.) in each vial, and thereactions stirred for 2 h at 20° C. The reactions were mixed and bufferexchanged using Sephadex G25 NAP-10 columns into PBS buffer, andconcentrated to a volume of 2.6 mL. Immediately after, an Ellman assaywas performed to give a Free Thiol to Antibody ratio (FTAR) of 5.6.

(b) Preparation of ADC 18

To the solution of thiol-activated Trastuzumab (500 μL, 5.0 mg, 33nmol), DMA was added (98.6 μL) followed by addition of a freshlyprepared solution of DL 9 (10 mM in DMA, 26.4 μL, 264 nmol, 8 eq.). Uponaddition of DL 9, the solution turned turbid. The conjugation reactionwas stirred for 2 h at 25° C. and purified by desalting using a SephadexG25 NAP-5 column into PBS buffer. The final target product ADC 18 wasconcentrated to a final concentration of 3.16 mg/mL as determined by UVand 390 μL (1.23 mg, 8.2 nmol, 25%) ADC solution was obtained.

Preparation of Antibody-Drug Conjugate ADC 19 with Traut's modifiedTrastuzumab and Compound DL 10 (a) Reaction of Trastuzumab with2-iminothiolane (Traut's reagent) to give thiol-activated Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (17.7 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (1.5 mL, 26.5 mg, 177 nmol) was diluted to aconcentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mMEDTA, pH 8) and fractionated in two vials (1.3 mL each). Traut's reagentwas added (14 mM, 61.8 μL, 866 nmol, 10 eq.) in each vial, and thereactions stirred for 2 h at 20° C. The reactions were mixed and bufferexchanged using Sephadex G25 NAP-10 columns into PBS buffer, andconcentrated to a volume of 2.6 mL. Immediately after, an Ellman assaywas performed to give a Free Thiol to Antibody ratio (FTAR) of 5.6.

(b) Preparation of ADC 19

To the solution of thiol-activated Trastuzumab (500 μL, 5.0 mg, 33nmol), DMA was added (98.6 μL) followed by addition of a freshlyprepared solution of DL 10 (10 mM in DMA, 26.4 μL, 264 nmol, 8 eq.).Upon addition of DL 10, the solution turned turbid. The conjugationreaction was stirred for 2 h at 25° C. and purified by desalting using aSephadex G25 NAP-5 column into PBS buffer. The final target product ADC19 was concentrated to a final concentration of 11.3 mg/mL as determinedby UV and 290 μL (3.2 mg, 21.3 nmol, 64%) ADC solution was obtained.

Preparation of Antibody-Drug Conjugate ADC 20 with Traut's modifiedTrastuzumab and Compound DL 11 (a) Reaction of Trastuzumab with2-iminothiolane (Traut's reagent) to give thiol-activated Trastuzumab

Trastuzumab (Trastuzumab purchased from Roche as a white lyophilisedpowder for the preparation of a concentrated solution for infusion) wasdissolved in 5 mL of phosphate buffer (50 mM, pH 8.0) and purified bydesalting using Sephadex G25 PD-10 columns into phosphate buffer (50 mM,pH 8.0). Concentration of Trastuzumab (17.7 mg/mL) was determined bymeasuring the absorbance at 280 nm.

Trastuzumab solution (1.5 mL, 26.5 mg, 177 nmol) was diluted to aconcentration of 10 mg/mL using phosphate buffer (50 mM phosphate, 2 mMEDTA, pH 8) and fractionated in two vials (1.3 mL each). Traut's reagentwas added (14 mM, 61.8 μL, 866 nmol, 10 eq.) in each vial, and thereactions stirred for 2 h at 20° C. The reactions were mixed and bufferexchanged using Sephadex G25 NAP-10 columns into PBS buffer, andconcentrated to a volume of 2.6 mL. Immediately after, an Ellman assaywas performed to give a Free Thiol to Antibody ratio (FTAR) of 5.6.

(b) Preparation of ADC 20

To the solution of thiol-activated Trastuzumab (500 μL, 5.0 mg, 33nmol), DMA was added (98.6 μL) followed by addition of a freshlyprepared solution of DL 11 (10 mM in DMA, 26.4 μL, 264 nmol, 8 eq.).Upon addition of DL 11, the solution turned turbid. The conjugationreaction was stirred for 2 h at 25° C. and purified by desalting using aSephadex G25 NAP-5 column into PBS buffer. The final target product ADC20 was concentrated to a final concentration of 3.73 mg/mL as determinedby UV and 440 μL (1.6 mg, 10.6 nmol, 32%) ADC solution was obtained.Example 3: Demonstrating the Cytotoxicity of the Antibody-DrugConjugates of the Present Invention

Bioassays for the Detection of Antitumor Activity

The aim of the assay was to evaluate the in vitro cytostatic (ability todelay or arrest tumor cell growth) or cytotoxic (ability to kill tumorcells) activity of the samples being tested.

Cell Lines and Cell Culture

The following human cell lines were obtained from the American TypeCulture Collection (ATCC): SK-BR-3 (ATCC HB-30), HCC-1954 (ATCCCRL-2338) (Breast cancer, HER2+); MDA-MB-231 (ATCC HTB-26) and MCF-7(ATCC HTB-22) (Breast cancer, HER2-), HT-1080 (ATCC CCL-121,fibrosarcoma, CD13+), Raji (ATCC CCL-86, Burkitt's lymphoma, CD13-) andRPMI 8226 (ATCC CRM-CCL-155, myeloma, CD13-). The human acutepromyelocytic leukemia cell line NB 4 (ACC 207, CD13+) was obtained fromthe Leibniz-Institut DSMZ (Braunschweig, Germany). Cells were maintainedat 37° C., 5% CO₂ and 95% humidity in Dulbecco's Modified Eagle's Medium(DMEM) (for SK-BR-3, MDA-MB-231 and MCF-7 cells), Eagle's MinimumEssential Medium (EMEM) (for HT-1080 cells) or RPMI-1640 (for the restof the cell lines), all media supplemented with 10% Fetal Calf Serum(FCS), 2 mM L-glutamine and 100 units/mL penicillin and streptomycin.

Cytotoxicity Assay

For SK-BR-3, HCC-1954, MDA-MB-231 and MCF-7 cells, a colorimetric assayusing Sulforhodamine B (SRB) was adapted for quantitative measurement ofcell growth and cytotoxicity, as described in V. Vichai and K.Kirtikara. Sulforhodamine B colorimetric assay for cytotoxicityscreening. Nature Protocols, 2006, 1, 1112-1116. Briefly, cells wereseeded in 96-well microtiter plates and allowed to stand for 24 hours indrug-free medium before treatment with vehicle alone or with the testedsubstances for 72 hours. For quantification, cells were washed twicewith phosphate buffered saline (PBS), fixed for 15 min in 1%glutaraldehyde solution, rinsed twice with PBS, stained in 0.4% (w/v)SRB with 1% (v/v) acetic acid solution for 30 min, rinsed several timeswith 1% acetic acid solution and air-dried. SRB was then extracted in 10mM Trizma base solution and the optical density measured at 490 nm in amicroplate spectrophotometer.

For HT-1080, NB 4, Raji and RPMI-8226 cells, an alternative colorimetricassay based on the reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wasused for quantitative measurement of cell viability as described by T.Mosmann in “Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays”, J ImmunolMethods, 1983, 65: 55-63. Briefly, cells were seeded in 96-well traysand treated as above and after 72 hours of exposure to the testedsubstances cellular viability was estimated from conversion of MTT(Sigma, St Louis, Mo., USA) to its coloured reaction product,MTT-formazan, which was dissolved in DMSO to measure its absorbance at540 nm in a microplate spectrophotometer.

Cell survival was expressed as percentage of control, untreated cellsurvival. All evaluations were performed in triplicate and the resultingdata were fitted by nonlinear regression to a four-parameters logisticcurve from which the IC50 value (the concentration of compound causing50% cell death as compared to the control cell survival) was calculated.

Bioactivity Example 1—Cytotoxicity of the Conjugate ADC 1 and RelatedReagents Against HER2 Positive and Negative Breast Cancer Cells

The in vitro cytotoxicity of the ADC 1 along with the parent cytotoxiccompounds DL 1 and 11-R and Trastuzumab were evaluated against fourdifferent human breast cancer cell lines over-expressing or not the HER2receptor, including SK-BR-3, HCC-1954 (HER2-positive cells) as well asMDA-MB-231 and MCF-7 (HER2-negative cells). Standard dose-response (DR)curves for 72 hours incubation with the tested substances wereperformed.

Cytotoxicity of Trastuzumab

The in vitro cytotoxicty of Trastuzumab was evaluated against thedifferent tumor cell lines by performing triplicate 10-points, 2.5-folddilution DR curves ranging from 50 to 0.01 μg/mL (3.33E-07-8.74E-11 M).Trastuzumab was completely inactive, not reaching the IC₅₀ in any of thecell lines tested, independently of their HER2 status as shown in Table10 where results corresponding to the geometric mean of the IC₅₀ valuesobtained in three independent experiments are presented.

TABLE 10 Summary of the in vitro cytotoxicity of Trastuzumab. HER2positive HER2 negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC₅₀,μg/mL >50 >50 >50 >50 IC₅₀, M >3.4E−07 >3.4E−07 >3.4E−07 >3.4E−07

Cytotoxicity of 11-R

The cytotoxicity of the intermediate compound 11-R was evaluated againstthe different tumor cell lines by performing triplicate 10-points,2.5-fold dilution DR curves ranging from 100 to 0.03 ng/mL(1.26E-07-3.3E-11 M).

As shown in Table 11, where results corresponding to the geometric meanof the IC₅₀ values obtained in three independent experiments arepresented, the cytotoxicity of this compound was similar in all thetumor cell lines regardless of their HER2 expression, with IC₅₀ valuesin the low nanomolar range, from 0.4 to 1.4 ng/mL (5.04E-10 to 1.70E-09M). The geometric mean IC₅₀ value across the whole cell panel was 0.79ng/mL (9.94E-10 M), with the standard geometric deviation being 1.8 inagreement with the homogeneity of results across the four cell lines.

TABLE 11 Summary of the in vitro cytotoxicity of 11-R HER2 positive HER2negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC₅₀, μg/mL 5.76E−04 1.35E−033.99E−04 1.24E−03 IC₅₀, M 7.27E−10 1.70E−09 5.04E−10 1.57E−09

Cytotoxicity of DL 1

The cytotoxicity of the intermediate compound DL 1 was evaluated againstthe different tumor cell lines by performing triplicate 10-points,2.5-fold dilution DR curves ranging from 10 μg/mL to 2.6 ng/mL(7.58E-06-1.99E-09 M).

As shown in Table 12, where results corresponding to the geometric meanof the IC₅₀ values obtained in three independent experiments arepresented, the cytotoxicity of this compound was similar in all thetumor cell lines regardless of their HER2 expression, with IC₅₀ valuesin the high nanomolar range, from 0.07 to 0.43 μg/mL (5.23E-08 to 3.11E-07 M). The geometric mean IC₅₀ value across the whole cell panel was0.16 μg/mL (1.15E-07 M), with the standard geometric deviation being 2.1in agreement with the homogeneity of results across the four cell lines.

TABLE 12 Summary of the in vitro cytotoxicity of DL 1 HER2 positive HER2negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC₅₀, μg/mL 1.16E−01 1.80E−017.28E−02 4.33E−01 IC₅₀, M 8.31E−08 1.29E−07 5.23E−08 3.11E−07

Cytotoxicity of ADC 1

The cytotoxicity of the ADC 1 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Theevaluation was performed in three independent experiments, FIG. 3 showsa representative DR curve corresponding to one of these experiments andTable 13 summarizes the results corresponding to the geometric mean ofthe IC₅₀ values obtained in the three independent experiments.

As observed in Table 13, ADC 1 showed a cytotoxicity which is similar tothat shown by the parent drug 11-R only in HER2-positive cells. However,in HER2-negative cells such toxicity is significantly lower: nearly40-fold lower according to the selectivity ratio obtained by dividingthe mean IC₅₀ value in HER2-negative cells between that in HER2-positivecells. This selectivity leads us to conclude that the conjugate isacting through the interaction of the antibody with the membraneassociated HER2 receptor on the tumor cells, followed by intracellulardelivery of the cytotoxic drug.

TABLE 13 Summary of the in vitro cytotoxicity of ADC 1 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 3.20E−011.38E+00 1.36E+01 4.90E+01 6.64E−01 2.58E01  38.8 IC₅₀ (M) 2.14E−099.20E−09 9.04E−08 3.27E−07 4.44E−09 1.72E−07

To graphically compare the cytotoxicity of the monoclonal antibodyTrastuzumab alone with that of the conjugate ADC 1, histograms showingthe percentages of cell survival after treatment of the different celllines with the monoclonal antibody alone (20 μg/mL) or ADC 1 at 16 or2.5 μg/mL, are shown in FIG. 4 . As observed, at similar concentrations(20 versus 16 μg/mL) Trastuzumab showed negligible cytotoxicityregardless the HER2 expression levels of the cell lines (cell survivalbetween 68% and 100%), whilst the ADC 1 showed a potentantiproliferative effect against the HER2-expressing cells HCC-1954 andSK-BR-3 (cell survival 23% and 8% respectively) and negligible againstthe HER2-negative cell line MCF-7 (100% cell survival). The effect ofthe ADC on MDA-MB-231 is noticeable at this concentration (22% cellsurvival) but it is very modest at lower concentrations (83% cellsurvival at 2.5 μg/mL) whereas it is still remarkable in theHER2-expressing cells (30% cell survival for both SK-BR-3 and HCC-1954).

These results clearly demonstrated the remarkable cytotoxicity andspecificity of the ADC 1 against HER2 expressing human tumor cells invitro.

Bioactivity Example 2—Cytotoxicity of the Conjugate ADC 2 and RelatedReagents Against HER2 Positive and Negative Breast Cancer Cells

The in vitro cytotoxicity of the ADC 2 along with the parent cytotoxiccompound DL 2 was evaluated against four different human breast cancercell lines over-expressing or not the HER2 receptor, including SK-BR-3,HCC-1954 (HER2 positive cells) as well as MDA-MB-231 and MCF-7 (HER2negative cells). Standard dose-response (DR) curves for 72 hoursincubation with the tested substances were performed. The results arealso compared with the parent cytotoxic compound 11-R and the monoclonalantibody Trastuzumab described above.

Cytotoxicity of DL 2

The cytotoxicity of the intermediate compound DL 2 was evaluated againstthe different tumor cell lines by performing triplicate 10-points,2.5-fold dilution DR curves ranging from 10 μg/mL to 2.6 ng/mL(6.26E-06-1.64E-09 M).

As shown in Table 14, the cytotoxicity of this compound was similar inall the tumor cell lines regardless of their HER2 expression, with IC50values in the sub-micromolar range, from 0.2 to 0.47 μg/mL (1.25E-07 to2.94E-07 M). The geometric mean IC₅₀ value across the whole cell panelwas 0.28 μg/mL (1.73E-07 M), with the standard geometric deviation being1.5 in agreement with the homogeneity of results across the four celllines.

TABLE 14 Summary of the in vitro cytotoxicity of DL 2 HER2 positive HER2negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC₅₀, μg/mL 2.00E−01 3.10E−012.00E−01 4.69E−01 IC₅₀, M 1.25E−07 1.94E−07 1.25E−07 2.94E−07

Cytotoxicity of ADC 2

The cytotoxicity of the ADC 2 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). FIG. 5shows the DR curve corresponding to this experiment and Table 15summarizes the IC₅₀ values obtained.

As observed in Table 15, ADC 2 showed higher cytotoxicity inHER2-positive cells than in HER2-negative cells: the ADC is nearly10-fold more potent in the former according to the selectivity ratioobtained by dividing the mean IC₅₀ value in HER2-negative cells betweenthat in HER2-positive cells. Although this selectivity is rather modest,it still leads us to conclude that the conjugate is acting through theinteraction of the antibody with the membrane associated HER2 receptoron the tumor cells, followed by intracellular delivery of the cytotoxicdrug.

TABLE 15 Summary of the in vitro cytotoxicity of ADC 2 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 2.00E+005.60E+00 1.70E+01 4.01E+01 3.34E+00 2.61E+01 7.8 IC₅₀ (M) 1.33E−083.73E−08 1.13E−07 2.67E−07 2.23E−08 1.74E−07

To graphically compare the cytotoxicity of the monoclonal antibodyTrastuzumab alone with that of the conjugate ADC 2, histograms showingthe percentages of cell survival after treatment of the different celllines with the monoclonal antibody alone (20 μg/mL) or ADC 2 at 16 or2.5 μg/mL, are shown in FIG. 6 . As observed, at similar concentrations(20 versus 16 μg/mL) Trastuzumab showed negligible cytotoxicitydisregarding the HER2 expression levels of the cell lines (cell survivalbetween 68% and 100%), whilst the ADC 2 showed a significantantiproliferative effect against the HER2-expressing cells HCC-1954 andSK-BR-3 (cell survival 31% and 40% respectively), such effect beingnegligible against the HER2-negative cell line MCF-7 (94% cellsurvival). The effect of the ADC on the HER2-negative cell lineMDA-MB-231 is also significant at this concentration, (58% cellsurvival) but it is null at lower concentrations (100% cell survival at2.5 μg/mL) whereas it is still remarkable in the HER2-expressing cells(63% cell survival for HCC-1954 and 51% for SK-BR-3).

Bioactivity Example 3—Cytotoxicity of the Conjugate ADC 3 and RelatedReagents Against HER2 Positive and Negative Breast Cancer Cells

The in vitro cytotoxicity of the ADC 3 along with the parent cytotoxiccompound DL 3 was evaluated against four different human breast cancercell lines over-expressing or not the HER2 receptor, including SK-BR-3,HCC-1954 (HER2 positive cells) as well as MDA-MB-231 and MCF-7 (HER2negative cells). Standard dose-response (DR) curves for 72 hoursincubation with the tested substances were performed. The results arealso compared with the parent cytotoxic compound 11-R and the monoclonalantibody Trastuzumab described above.

Cytotoxicity of DL 3

The cytotoxicity of the intermediate compound DL 3 was evaluated againstthe different tumor cell lines by performing triplicate 10-points,2.5-fold dilution DR curves ranging from 10 μg/mL to 2.6 ng/mL(6.62E-06-1.74E-09 M).

As shown in Table 16, the cytotoxicity of this compound was similar inall the tumor cell lines regardless of their HER2 expression, with IC50values in the sub-micromolar range, from 0.15 to 0.28 μg/mL (9.93E-08 to1.85E-07 M). The geometric mean IC₅₀ value across the whole cell panelwas 0.2 μg/mL (1.33E-07 M), with the standard geometric deviation being1.4 in agreement with the homogeneity of results across the four celllines.

TABLE 16 Summary of the in vitro cytotoxicity of DL 3 HER2 positive HER2negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC₅₀, μg/mL 1.60E−01 2.40E−011.50E−01 2.79E−01 IC₅₀, M 1.06E−07 1.59E−07 9.93E−08 1.85E−07

Cytotoxicity of ADC 3

The cytotoxicity of the ADC 3 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 50 μg/mL to 13 ng/mL (3.33E-07-8.74E-11 M). FIG. 7shows the DR curve corresponding to this experiment and Table 17summarizes the IC₅₀ values obtained.

As observed in Table 17, ADC 3 showed a cytotoxicity which only inHER2-positive cells is similar to that shown by the parent drug 11-R.However, in HER2-negative cells such toxicity is significantly lower:23-fold lower according to the selectivity ratio obtained by dividingthe mean IC₅₀ value in HER2-negative cells between that in HER2-positivecells. This selectivity leads us to conclude that the conjugate isacting through the interaction of the antibody with the membraneassociated HER2 receptor on the tumor cells, followed by intracellulardelivery of the cytotoxic drug as previously stated for ADC 1 and ADC 2.

TABLE 17 Summary of the in vitro cytotoxicity of ADC 3 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 6.71E−018.10E−01 1.30E+01 2.21E+01 7.37E−01 1.70E+01 23.0 IC₅₀ (M) 4.47E−095.40E−09 8.67E−08 1.47E−07 4.91E−09 1.13E−07

To graphically compare the cytotoxicity of the monoclonal antibodyTrastuzumab alone with that of the conjugate ADC 3, histograms showingthe percentages of cell survival after treatment of the different celllines with the monoclonal antibody alone (20 μg/mL) or ADC 3 at 20 or 3μg/mL, are shown in FIG. 8 . As observed, at 20 μg/mL Trastuzumab showednegligible cytotoxicity regardless the HER2 expression levels of thecell lines (cell survival between 68% and 100%), whilst the ADC 3 showeda potent antiproliferative effect against the HER2-expressing cellsHCC-1954 and SK-BR-3 (cell survival 23% and 32% respectively) and onlymarginal against the HER2-negative cell line MCF-7 (88% cell survival).The effect of the ADC on MDA-MB-231 is noticeable at this concentration(32% cell survival) but it is negligible at lower concentrations (91%cell survival at 3 μg/mL) whereas it is still remarkable in theHER2-expressing cells (34% cell survival for HCC-1954 and 45% forSK-BR-3).

Bioactivity Example 4—Cytotoxicity of the Conjugate ADC 4 Against HER2Positive and Negative Breast Cancer Cells

The in vitro cytotoxicity of the ADC ADC 4 was evaluated against fourdifferent human breast cancer cell lines over-expressing or not the HER2receptor, including SK-BR-3, HCC-1954 (HER2 positive cells) as well asMDA-MB-231 and MCF-7 (HER2 negative cells). Standard dose-response (DR)curves for 72 hours incubation with the ADC were performed. The resultsare also compared with the parent cytotoxic compounds 11-R and DL 1 aswell as the monoclonal antibody Trastuzumab described above.

Cytotoxicity of ADC 4

The cytotoxicity of the ADC 4 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 50 μg/mL to 13 ng/mL (3.33E-07-8.74E-11 M). FIG. 9shows the DR curve corresponding to this experiment and Table 18summarizes the IC50 values obtained.

As observed in Table 18, ADC 4 showed a cytotoxicity which only inHER2-positive cells is similar to that shown by the parent drug 11-R.However, in HER2-negative cells such cytotoxicity is nearly 100-foldlower according to the selectivity ratio obtained by dividing the meanIC₅₀ value in HER2-negative cells between that in HER2-positive cells.As already stated for ADC 1, such selectivity leads us to conclude thatthe conjugate is acting through the interaction of the antibody with themembrane associated HER2 receptor on the tumor cells, followed byintracellular delivery of the cytotoxic drug. Moreover, these resultsobtained with ADC 4 demonstrate that the antitumoral potential endowedby the payload is similar regardless of the antibody residue involved inthe covalent bond, either Cys or Lys.

TABLE 18 Summary of the in vitro cytotoxicity of ADC 4 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 3.20E−021.50E−01 3.50E+00 8.10E+00 6.92E−02 5.32E+00 76.86 IC₅₀ (M) 2.13E−101.00E−09 2.33E−08 5.40E−08 4.62E−10 3.55E−08

To graphically compare the cytotoxicity of the monoclonal antibodyTrastuzumab alone with that of the conjugate ADC 4, histograms showingthe percentages of cell survival after treatment of the different celllines with the monoclonal antibody alone at 3 μg/mL or with the ADC at 3or 0.2 μg/mL, are shown in FIG. 10 . As observed, at 3 μg/mL Trastuzumabshowed scarce cytotoxicity irrespective of the HER2 expression levels ofthe cell lines (cell survival between 60% and 90%), whilst the ADC 4showed a potent antiproliferative effect against the HER2-expressingcells HCC-1954 and SK-BR-3 (cell survival 11% and 23% respectively) andnearly no effect on the HER2-negative cell line MCF-7. The effect of theADC on the cell line MDA-MB-231 is noticeable at this concentration (55%cell survival) but it is negligible at lower concentrations (97-100%cell survival at 0.2 μg/mL) whereas it is clearly remarkable in theHER2-expressing cells (38% cell survival for HCC-1954 and 26% forSK-BR-3).

Bioactivity Example 5—Cytotoxicity of the Conjugate ADC 6 and RelatedReagents Against Her2 Positive and Negative Breast Cancer Cells

The in vitro cytotoxicity of the ADC 6 along with the parent cytotoxiccompounds DL 5 and 12 R and were evaluated against four different humanbreast cancer cell lines over-expressing or not the HER2 receptor,including SK-BR-3, HCC-1954 (HER2-positive cells) as well as MDA-MB-231and MCF-7 (HER2-negative cells). Standard dose-response (DR) curves for72 hours incubation with the tested substances were performed.

Cytotoxicity of 12-R

The cytotoxicity of the intermediate compound 12-R was evaluated againstthe different tumor cell lines by performing triplicate 10-points,2.5-fold dilution DR curves ranging from 100 to 0.03 ng/mL(1.28E-07-3.83E-11 M).

As shown in Table 19, where results corresponding to the geometric meanof the IC50 values obtained in three independent experiments arepresented, the cytotoxicity of this compound was similar in all thetumor cell lines regardless of their HER2 expression, with IC₅₀ valuesin the low nanomolar range, from 0.4 to 1.3 ng/mL (5.62E-10 to 1.62E-09M). The geometric mean IC₅₀ value across the whole cell panel was 0.71ng/mL (9.06E-10 M), with the standard geometric deviation being 1.7 inagreement with the homogeneity of results across the four cell lines.

TABLE 19 Summary of the in vitro cytotoxicity of 12-R HER2 positive HER2negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC₅₀, μg/mL 4.41E−04 1.27E−034.93E−04 9.24E−04 IC₅₀, M 5.62E−10 1.62E−09 6.29E−10 1.18E−09

Cytotoxicity of DL 5

The cytotoxicity of the intermediate compound DL 5 was evaluated againstthe different tumor cell lines by performing triplicate 10-points,2.5-fold dilution DR curves ranging from 10 μg/mL to 2.6 ng/mL(6.66E-06-1.73E-09 M).

As shown in Table 20, the cytotoxicity of this compound was similar inall the tumor cell lines regardless of their HER2 expression, with IC₅₀values in the high nanomolar range, from 0.10 to 0.19 μg/mL (6.53E-08 to1.27E-07 M). The geometric mean IC₅₀ value across the whole cell panelwas 0.13 μg/mL (8.84E-08 M), with the standard geometric deviation being1.4 in agreement with the homogeneity of results across the four celllines.

TABLE 20 Summary of the in vitro cytotoxicity of DL 5 HER2 positive HER2negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC₅₀, μg/mL 9.81E−02 1.91E−019.81E−02 1.70E−01 IC₅₀, M 6.53E−08 1.27E−07 6.53E−08 1.13E−07

Cytotoxicity of ADC 6

The cytotoxicity of the ADC 6 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). FIG. 11shows the DR curve obtained and Table 21 summarizes the deduced IC₅₀values.

As observed in Table 21, the cytotoxicity of ADC 6 is comparable to thatshown by the parent drug 12-R only in HER2-positive cells. However, inHER2-negative cells such toxicity is lower: nearly 10-fold loweraccording to the selectivity ratio obtained by dividing the mean IC₅₀value in HER2-negative cells between that in HER2-positive cells. Thesedata lead us to conclude that the conjugate is acting through theinteraction of the antibody with the membrane associated HER2 receptoron the tumor cells, followed by intracellular delivery of the cytotoxicdrug.

TABLE 21 Summary of the in vitro cytotoxicity of ADC 6 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 6.50E−014.10E+00 1.20E+01 1.70E+01 1.63E00  1.43E01  8.8 IC₅₀ (M) 4.33E−092.73E−08 8.00E−08 1.13E−07 1.08E−08 9.51E−08

Bioactivity Example 6—Cytotoxicity of the Conjugate ADC 11 and RelatedReagents Against Her2 Positive and Negative Breast Cancer Cells

The in vitro cytotoxicity of the ADC 11 along with the parent cytotoxiccompounds DL 6 and 12 R and were evaluated against four different humanbreast cancer cell lines over-expressing or not the HER2 receptor,including SK-BR-3, HCC-1954 (HER2-positive cells) as well as MDA-MB-231and MCF-7 (HER2-negative cells). Standard dose-response (DR) curves for72 hours incubation with the tested substances were performed.

Cytotoxicity of DL 6

The cytotoxicity of the intermediate compound DL 6 was evaluated againstthe different tumor cell lines by performing triplicated 10-points,2.5-fold dilution DR curves ranging from 1 μg/mL to 0.26 ng/mL(7.23E-07-1.88E-10 M).

As shown in Table 22, the cytotoxicity of this compound was similar inall the tumor cell lines regardless of their HER2 expression, with IC50values in the high nanomolar range, from 0.04 to 0.3 μg/mL (3.1 E-08 to1.97E-07 M). The geometric mean IC₅₀ value across the whole cell panelwas 0.11 μg/mL (7.89E-08 M), with the standard geometric deviation being2.31 in agreement with the homogeneity of results across the four celllines.

TABLE 22 Summary of the in vitro cytotoxicity of DL 6 HER2 positive HER2negative SK-BR-3 HCC-1954 MDA-MB-231 MCF-7 IC₅₀, μg/mL 7.09E−02 1.71E−014.29E−02 2.73E−01 IC₅₀, M 5.13E−08 1.24E−07 3.10E−08 1.97E−07

Cytotoxicity of ADC 11

The cytotoxicity of the ADC 11 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 50 μg/mL to 13 ng/mL (3.33E-07-8.74E-11 M). Table 23summarizes the deduced IC₅₀ values.

TABLE 23 Summary of the in vitro cytotoxicity of ADC 11 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 1.10E+013.60E+01 >5.00E+01 >5.00E+01 1.99E+01 >5.00E+01 >2.5 IC₅₀ (M) 7.33E−082.40E−07 >3.33E−07 >3.33E−07 1.33E−07 >3.33E−07

Bioactivity Example 7—Cytotoxicity of the Conjugate ADC 12 Against Her2Positive and Negative Breast Cancer Cells

The in vitro cytotoxicity of the ADC 12 was evaluated against fourdifferent human breast cancer cell lines over-expressing or not the HER2receptor, including SK-BR-3, HCC-1954 (HER2-positive cells) as well asMDA-MB-231 and MCF-7 (HER2-negative cells). Standard dose-response (DR)curves for 72 hours incubation with the tested substances wereperformed.

The cytotoxicity of the ADC 12 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table24 summarizes the deduced IC50 values. As observed, ADC 12 issignificantly more active in in HER2-positive cells, yielding anoticeable selectivity ratio higher than 20.

TABLE 24 Summary of the in vitro cytotoxicity of ADC 12 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 1.80E−012.90E−01 1.91E+00 1.30E+01 2.28E−01 4.98E+00 21.8 IC₅₀ (M) 1.20E−091.93E−09 1.27E−08 8.67E−08 1.52E−09 3.32E−08

Bioactivity Example 8—Cytotoxicity of the Conjugate ADC 13 Against Her2Positive and Negative Breast Cancer Cells

The cytotoxicity of the ADC 13 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table25 summarizes the deduced IC₅₀ values. As observed, ADC 13 issignificantly more active in HER2-positive cells, yielding anoutstanding selectivity ratio close to 100.

TABLE 25 Summary of the in vitro cytotoxicity of ADC 13 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 1.50E−012.60E−01 1.10E+01 2.90E+01 1.97E−01 1.78E+01 90.43 IC₅₀ (M) 1.00E−091.73E−09 7.33E−08 1.93E−07 1.32E−09 1.19E−07

Bioactivity Example 9—Cytotoxicity of the Conjugate ADC 14 Against Her2Positive and Negative Breast Cancer Cells

The cytotoxicity of the ADC 14 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table26 summarizes the deduced IC50 values. Remarkably, ADC 14 has shown acytotoxic activity in the low nM range comparable to that of theparental drug 11R but only in HER2-expressing cells, whilst it hasfailed to show any activity in HER2-negative cells within the range ofconcentrations tested, thus confirming an outstanding selectivity.

TABLE 26 Summary of the in vitro cytotoxicity of ADC 14 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 1.10E−011.80E−01 >2.51E+01 >2.51E+01 1.41E−01 >2.51E+01 >178 IC₅₀ (M) 7.33E−101.20E−09 >1.67E−07 >1.67E−07 9.38E−10 >1.67E−07

Bioactivity Example 10—Cytotoxicity of the Conjugate ADC 15 Against Her2Positive and Negative Breast Cancer Cells

The cytotoxicity of the ADC 15 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 50 μg/mL to 13 ng/mL (3.33E-07-8.74E-11 M). Table 27summarizes the deduced IC₅₀ values. As observed, ADC 15 is significantlymore active in in HER2-positive cells, yielding an outstandingselectivity ratio close to 200.

TABLE 27 Summary of the in vitro cytotoxicity of ADC 15 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 8.00E−021.80E−01 2.10E+01 2.21E+01 1.20E−01 2.15E+01 179.4 IC₅₀ (M) 5.33E−101.20E−09 1.40E−07 1.47E−07 8.00E−10 1.43E−07

Bioactivity Example 11—Cytotoxicity of the Conjugate ADC 16 Against Her2Positive and Negative Breast Cancer Cells

The cytotoxicity of the ADC 16 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table28 summarizes the deduced IC₅₀ values. As observed, ADC 16 issignificantly more active in in HER2-positive cells, yielding anoutstanding selectivity ratio above 100.

TABLE 28 Summary of the in vitro cytotoxicity of ADC 16 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 4.01E−028.00E−01 1.30E+01 2.70E+01 1.79E−01 1.87E+01 104.7 IC₅₀ (M) 2.67E−105.33E−09 8.67E−08 1.80E−07 1.19E−09 1.25E−07

Bioactivity Example 12—Cytotoxicity of the Conjugate ADC 17 Against Her2Positive and Negative Breast Cancer Cells

The cytotoxicity of the ADC 17 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table29 summarizes the deduced IC₅₀ values.

TABLE 29 Summary of the in vitro cytotoxicity of ADC 17 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 7.40E+001.40E+01 4.40E+01 4.20E+01 1.02E+01 4.30E+01 4.22 IC₅₀ (M) 4.93E−089.33E−08 2.93E−07 2.80E−07 6.78E−08 2.86E−07

Bioactivity Example 13—Cytotoxicity of the Conjugate ADC 18 Against Her2Positive and Negative Breast Cancer Cells

The cytotoxicity of the ADC 18 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table30 summarizes the deduced IC₅₀ values.

TABLE 30 Summary of the in vitro cytotoxicity of ADC 18 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 4.40E+002.21E+01 4.31E+01 9.71E+01 9.84E+00 6.46E+01 6.57 IC₅₀ (M) 2.93E−081.47E−07 2.87E−07 6.47E−07 6.56E−08 4.31E−07

Bioactivity Example 14—Cytotoxicity of the Conjugate ADC 19 Against Her2Positive and Negative Breast Cancer Cells

The cytotoxicity of the ADC 19 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table31 summarizes the deduced IC₅₀ values.

TABLE 31 Summary of the in vitro cytotoxicity of ADC 19 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 8.30E+003.41E+01 >1.00E+02 >1.00E+02 1.68E+01 >1.00E+02 >5.95 IC₅₀ (M) 5.53E−082.27E−07 >6.67E−07 >6.67E−07 1.12E−07 >6.67E−07

Bioactivity Example 15—Cytotoxicity of the Conjugate ADC 20 Against Her2Positive and Negative Breast Cancer Cells

The cytotoxicity of the ADC 20 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table32 summarizes the deduced IC₅₀ values.

TABLE 32 Summary of the in vitro cytotoxicity of ADC 20 IC50 in IC50 inHER2+ HER2− HER2 positive HER2 negative (geom. (geom. Selec. SK-BR-3HCC-1954 MDA-MB-231 MCF-7 mean) mean ratio IC₅₀ (μg/mL) 2.21E+001.61E+01 5.40E+01 4.01E+01 5.95E+00 4.65E+01 7.82 IC₅₀ (M) 1.47E−081.07E−07 3.60E−07 2.67E−07 3.97E−08 3.10E−07

Bioactivity Example 16—Cytotoxicity of the Conjugate ADC 7 and RelatedReagents Against CD13 Positive and Negative Cancer Cells Cytotoxicity of11-R

The cytotoxicity of the intermediate compound 11-R was evaluated againstthe different tumor cell lines by performing triplicate 10-points,2.5-fold dilution DR curves ranging from 100 μg/mL to 0.03 ng/mL(1.26E-07-3.3E-11 M).

As shown in Table 33, where results corresponding to the geometric meanof the IC50 values obtained in three different experiments arepresented, the cytotoxicity of this compound was similar in all thetumor cell lines regardless of their CD13 expression, with IC₅₀ valuesin the low nanomolar range, from 0.5 to 1.2 ng/mL (5.8E-10 to 1.51 E-09M). The geometric mean IC₅₀ value across the whole cell panel was 0.84ng/mL (1.06E-09 M) with the standard geometric deviation being 1.5 inagreement with the homogeneity of results across the four cell lines.

TABLE 33 Summary of the in vitro cytotoxicity of 11-R CD13 positive CD13negative HT-1080 NB-4 Raji RPMI8226 IC₅₀, μg/mL 1.20E−03 4.60E−049.67E−04 9.28E−04 IC₅₀, M 1.51E−09 5.80E−10 1.22E−09 1.17E−09

Cytotoxicity of ADC 7

The cytotoxicity of the ADC 7 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table34 summarizes the deduced IC₅₀ values. Remarkably, ADC 7 has shown acytotoxic activity in the low nM range comparable to that of theparental drug 11-R but only in CD13-expressing cells, whereas itsactivity in CD13-negative cells is rather modest. Consequently, anoutstanding selectivity fair above 100 can be observed as a function ofCD13 expression.

TABLE 34 Summary of the in vitro cytotoxicity of ADC 7 IC50 in IC50 inCD13+ CD13− CD13 positive CD13 negative (geom. (geom. Selec. HT-1080NB-4 Raji RPMI8226 mean) mean ratio IC₅₀ (μg/mL) 1.30E−01 2.70E−012.21E+01 4.10E+01 1.87E−01 3.00E+01 160.4 IC₅₀ (M) 8.67E−10 1.80E−091.47E−07 2.73E−07 1.25E−09 2.00E−07

Bioactivity Example 17—Cytotoxicity of the Conjugate ADC 8 Against CD13Positive and Negative Cancer Cells

The cytotoxicity of the ADC 8 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table35 summarizes the deduced IC50 values. ADC 8 has shown a cytotoxicactivity in the low nM range comparable to that of the parental drug11-R but only in CD13-expressing cells, whereas its activity inCD13-negative cells is rather modest. Consequently, an outstandingselectivity close to 200 was obtained as a function of CD13 expression.

TABLE 35 Summary of the in vitro cytotoxicity of ADC 8 IC50 in IC50 inCD13+ CD13− CD13 positive CD13 negative (geom. (geom. Selec. HT-1080NB-4 Raji RPMI8226 mean) Mean) ratio IC₅₀ (μg/mL) 1.61E−01 2.51E−014.31E+01 3.30E+01 2.01E−01 3.77E+01 188 IC₅₀ (M) 1.07E−09 1.67E−092.87E−07 2.20E−07 1.34E−09 2.51E−07

Bioactivity Example 18—Cytotoxicity of the Conjugate ADC 9 and RelatedReagents Against Cd13 Positive and Negative Cancer Cells Cytotoxicity of12-R

The cytotoxicity of the intermediate compound 12-R was evaluated againstthe different tumor cell lines by performing triplicate 10-points,2.5-fold dilution DR curves ranging from 100 to 0.03 ng/mL(1.28E-07-3.83E-11 M).

As shown in Table 36, where results corresponding to the geometric meanof the IC₅₀ values obtained in three independent experiments arepresented, the cytotoxicity of this compound was similar in all thetumor cell lines regardless of their CD13 expression, with IC₅₀ valuesin the low nanomolar range, from 0.3 to 1.1 ng/mL (4.21E-10 to 1.40E-09M). The geometric mean IC₅₀ value across the whole cell panel was 0.6ng/mL (7.8E-10 M), with the standard geometric deviation being 1.7 inagreement with the homogeneity of results across the four cell lines.

TABLE 36 Summary of the in vitro cytotoxicity of 12-R. CD13 positiveCD13 negative HT-1080 NB-4 Raji RPMI8226 IC₅₀, μg/mL 1.10E−03 3.30E−044.70E−04 8.30E−04 IC₅₀, M 1.40E−09 4.21E−10 6.00E−10 1.06E−09

Cytotoxicity of ADC-9

The cytotoxicity of the ADC 9 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table37 summarizes the deduced IC₅₀ values. ADC 9 has shown a cytotoxicactivity in the low nM range comparable to that of the parental drug12-R but only in CD13-expressing cells, whereas its activity inCD13-negative cells is rather modest. Consequently, an outstandingselectivity ratio above 200 was yielded as a function of CD13expression.

TABLE 37 Summary of the in vitro cytotoxicity of ADC 9 IC50 in IC50 inCD13+ CD13− CD13 positive CD13 negative (geom. (geom. Selec. HT-1080NB-4 Raji RPMI8226 mean) Mean) ratio IC₅₀ (μg/mL) 1.20E−01 5.21E−021.70E+01 1.70E+01 7.90E−02 1.70E+01 214 IC₅₀ (M) 8.00E−10 3.47E−101.13E−07 1.13E−07 5.27E−10 1.13E−07

Bioactivity Example 19—Cytotoxicity of the Conjugate ADC 10 Against CD13Positive and Negative Cancer Cells

The cytotoxicity of the ADC 10 was evaluated against the different tumorcell lines by performing triplicate 10-points, 2.5-fold dilution DRcurves ranging from 100 μg/mL to 26 ng/mL (6.67E-07-1.75E-10 M). Table38 summarizes the deduced IC₅₀ values. ADC 10 has shown a cytotoxicactivity in the low nM range comparable to that of the parental drug11-R but only in CD13-expressing cells, whereas its activity inCD13-negative cells is rather modest. Consequently, a remarkableselectivity close to 100 was obtained as a function of CD13 expression.

TABLE 38 Summary of the in vitro cytotoxicity of ADC 10 IC50 in IC50 inCD13+ CD13− CD13 positive CD13 negative (geom. (geom. Selec. HT1080 NB-4Raji RPMI 8226 mean) mean ratio IC₅₀ (μg/mL) 9.71E−02 9.71E−01 3.30E+012.81E+01 3.07E−01 3.04E+01 99.14 IC₅₀ (M) 6.47E−10 6.47E−09 2.20E−071.87E−07 2.05E−09 2.03E−07

Example 4: Demonstrating the In Vivo Efficacy of the Antibody-DrugConjugates of the Present Invention

Trastuzumab-based antibody drug conjugate ADC 1 was tested in several invivo models. ADC-1 batches used in these studies were prepared using 2.2eq of TCEP (ADC 1 2.2 TCEP) or 3 eq of TCEP (ADC-1), These batches weremanufactured using the procedures described above with the exceptionthat the final purification by size exclusion chromatography (SEC) wascarried out using a Hi Load 26/600 Superdex™ 200 μg column and PBS (pH7.4) as eluent.

ADC-1 and ADC 1 2.2 TCEP were evaluated in a breast HER2 positive model,BT-474 together with their payload, compound 11-R. Of note, in spite ofthe low dose (suboptimal) administered to mice bearing tumors in thisexperiment, encouraging positive result was obtained (See FIG. 12 ).Therefore, a set of new experiments, aimed at evaluating the antitumoractivity in breast and non-breast Her2 positive models were performed.ADC-1 was evaluated at a higher dose level in the breast tumor model,JIMT-1 (See FIG. 13 ), in one ovarian tumor model, SK-OV-3 (FIG. 14 )and in a gastric model, Gastric-008, a patient derived xenograft (PDX),(FIG. 16 ). Additionally ADC-1 2.2 TCEP) was evaluated in anothergastric model, N87 (FIG. 15 ).

Briefly, 4 to 6 week-old athymic nu/nu (N87, Gastric-008 or SK-OV-3) orSCID (BT-474 or JIMT-1) mice were subcutaneously implanted with eithertumor cell suspension (JIMT-1 or N87) or tumor fragments (BT-474,Gastric-008 or SK-OV3) previously generated in donor mice.

Tumor dimension and body weight was recorded 3 times per week startingfrom the first day of treatment (Day 0). Treatments producing >20%lethality and/or 20% net body weight loss were considered toxic. Tumorvolume was calculated using the equation (a·b²)/2, where a and b werethe longest and shortest diameters, respectively. Animals wereeuthanized when their tumors reached ca. 2,000 mm³ and/or severenecrosis was seen. Median was calculated for tumor volume on eachmeasurement day. Complete tumor regression (CR) was defined when tumorvolume <63 mm³ for 2 or more consecutive measurements.

The animals were implanted as above described and when tumors reachedca. 150-200 mm³, tumor bearing animals (N=8-10/group) were randomlyallocated into treatment groups according to the following experimentaldesign:

Tumor Group Dose (mg/kg) JIMT-1 Control 0.0 11-R 5.0 ADC-1 30.0 ADC-110.0 ADC-1 5.0 N87 Control 0.0 11-R 5.0 ADC-1 (2.2 TCEP) 30.0 ADC-1 (2.2TCEP) 10.0 ADC-1 (2.2 TCEP) 5.0 SK-OV-3 Control 0.0 11-R 5.0 ADC-1 30.0ADC-1 10.0 ADC-1 5.0 Gastric-008 Control 0.0 11-R 5.0 ADC-1 30.0 ADC-110.0 ADC-1 5.0 BT-474 Control 0.0 11-R 5.0 ADC-1 (2.2 TCEP) 6.5 ADC-12.24 ADC-1 (2.2 TCEP) 1.6

1. A drug conjugate comprising a drug moiety covalently attached to therest of the drug conjugate, the drug conjugate having formula[D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab wherein: D is a drug moietyhaving the following formula (I) or a pharmaceutically acceptable salt,ester, solvate, tautomer or stereoisomer thereof,

wherein: D is covalently attached via a hydroxy or amine group to(X)_(b) if any, or (AA)_(w) if any, or to (T)_(g) if any, or (L); Y is—NH— or —O—; R₁ is —OH or —CN; R₂ is a —C(═O)R_(a) group; R₃ is hydrogenor a —OR_(b) group; R₄ is selected from —CH₂O— and —CH₂NH—; R_(a) isselected from hydrogen, substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl; R_(b) is selected from substituted orunsubstituted C₁-C₁₂ alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl,and substituted or unsubstituted C₂-C₁₂ alkynyl; X and T are extendinggroups that may be the same or different; each AA is independently anamino acid unit; L is a linker group; w is an integer ranging from 0 to12; b is an integer of 0 or 1; g is an integer of 0 or 1; Ab is a moietycomprising at least one antigen binding site; and n is the ratio of thegroup [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] to the moiety comprising atleast one antigen binding site and is in the range from 1 to 20; whereinthe drug moiety D is conjugated via the R₄ position.
 2. The drugconjugate according to claim 1, wherein D is selected from formula Ia orIb, or a pharmaceutically acceptable salt or ester thereof:

wherein: Y is —NH— or —O—; R₁ is —OH or —CN; R₂ is a —C(═O) R_(a) group;R₃ is hydrogen or a —OR_(b) group; R₄ is selected from —CH₂O— and—CH₂NH—; R_(a) is selected from hydrogen, substituted or unsubstitutedC₁-C₁₂ alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl, andsubstituted or unsubstituted C₂-C₁₂ alkynyl; and R_(b) is selected fromsubstituted or unsubstituted C₁-C₁₂ alkyl, substituted or unsubstitutedC₂-C₁₂ alkenyl, and substituted or unsubstituted C₂-C₁₂ alkynyl.
 3. Thedrug conjugate according to claim 1 or claim 2, wherein R₄ is —CH₂O-. 4.The drug conjugate according to claim 1 or claim 2, wherein R₄ is—CH₂NH—.
 5. The drug conjugate according to claim 1, wherein D is acompound of formula:

or a pharmaceutically acceptable salt or ester thereof; wherein the wavyline indicates the point of covalent attachment to (X)_(b) if any, or(AA)_(w) if any, or to (T)_(g) if any, or (L); preferably of formula:

or a pharmaceutically acceptable salt or ester thereof; preferably offormula:

or a pharmaceutically acceptable salt or ester thereof.
 6. The drugconjugate according to claim 1, wherein D is a compound of formula:

or a pharmaceutically acceptable salt or ester thereof; wherein the wavyline indicates the point of covalent attachment to (X)_(b) if any, or(AA)_(w) if any, or to (T)_(g) if any, or (L).
 7. The drug conjugateaccording to claim 1, wherein D is a compound of formula:

or a pharmaceutically acceptable salt or ester thereof; wherein the wavyline indicates the point of covalent attachment to (X)_(b) if any, or(AA)_(w) if any, or to (T)_(g) if any, or (L).
 8. The drug conjugateaccording to claim 1, wherein D is a compound of formula:

or a pharmaceutically acceptable salt or ester thereof; wherein the wavyline indicates the point of covalent attachment to (X)_(b) if any, or(AA)_(w) if any, or to (T)_(g) if any, or (L).
 9. The drug conjugateaccording to claim 1, wherein D is a compound of formula:

or a pharmaceutically acceptable salt or ester thereof; wherein the wavyline indicates the point of covalent attachment to (X)_(b) if any, or(AA)_(w) if any, or to (T)_(g) if any, or (L).
 10. The drug conjugateaccording to claim 1, wherein D is a compound of formula:

or a pharmaceutically acceptable salt or ester thereof; wherein the wavyline indicates the point of covalent attachment to (X)_(b) if any, or(AA)_(w) if any, or to (T)_(g) if any, or (L).
 11. A drug conjugatecomprising a drug moiety covalently attached to the rest of the drugconjugate, the drug conjugate having formula[D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]_(n)-Ab wherein: D is a drug moietyhaving the following formula (IH) or a pharmaceutically acceptable salt,ester, solvate, tautomer or stereoisomer thereof:

wherein: the wavy line indicates the point of covalent attachment to(X)_(b) if any, or (AA)_(w) if any, or to (T)_(g) if any, or (L); each Yand Z is independently selected from —NH— and —O—; R₁ is —OH or —CN; R₂is a —C(═O)R_(a) group; R₃ is hydrogen or a —OR_(b) group; R_(a) isselected from hydrogen, substituted or unsubstituted C₁-C₁₂ alkyl,substituted or unsubstituted C₂-C₁₂ alkenyl, and substituted orunsubstituted C₂-C₁₂ alkynyl, wherein the optional substituents are oneor more substituents R_(x); and R_(b) is selected from substituted orunsubstituted C₁-C₁₂ alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl,and substituted or unsubstituted C₂-C₁₂ alkynyl, wherein the optionalsubstituents are one or more substituents R_(x); substituents R_(x) areselected from the group consisting of C₁-C₁₂ alkyl groups which may beoptionally substituted with at least one group R_(y), C₂-C₁₂ alkenylgroups which may be optionally substituted with at least one groupR_(y), C₂-C₁₂ alkynyl groups which may be optionally substituted with atleast one group R_(y), halogen atoms, oxo groups, thio groups, cyanogroups, nitro groups, OR_(y), OCOR_(y), OCOOR_(y), COR_(y), COOR_(y),OCONR_(y)R_(z), CONR_(y)R_(z), S(O)R_(y), SO₂R_(y), P(O)(R_(y))OR_(z),NR_(y)R_(z), NR_(y)COR_(z), NR_(y)C(═O)NR_(y)R_(z),NR_(y)C(═NR_(y))NR_(y)R_(z), aryl groups having from 6 to 18 carbonatoms in one or more rings which may optionally be substituted with oneor more substituents which may be the same or different selected fromthe group consisting of R_(y), OR_(y), OCOR_(y), OCOOR_(y), NR_(y)R_(z),NR_(y)COR_(z), and NR_(y)C(═NR_(y))NR_(y)R_(z), aralkyl groupscomprising an alkyl group having from 1 to 12 carbon atoms substitutedwith an optionally substituted aryl group as defined above, aralkyloxygroups comprising an alkoxy group having from 1 to 12 carbon atomssubstituted with an optionally substituted aryl group as defined above,and a 5-to 14-membered saturated or unsaturated heterocyclic grouphaving one or more rings and comprising at least one oxygen, nitrogen orsulphur atom in said ring(s), said heterocyclic group optionally beingsubstituted with one or more substituents R_(y), and where there is morethan one optional substituents on any given group the optionalsubstituents R_(y) may be the same or different; each R_(y) and R_(z) isindependently selected from the group consisting of hydrogen, C₁-C₁₂alkyl groups, C₁-C₁₂ alkyl groups that are substituted with at least onehalogen atom, aralkyl groups comprising a C₁-C₁₂ alkyl group that issubstituted with an aryl group having from 6 to 18 carbon atoms in oneor more rings and heterocycloalkyl groups comprising a C₁-C₁₂ alkylgroup that is substituted with a 5- to 14-membered saturated orunsaturated heterocyclic group having one or more rings and comprisingat least one oxygen, nitrogen or sulphur atom in said ring(s); X and Tare extending groups that may be the same or different; each AA isindependently an amino acid unit; L is a linker group; w is an integerranging from 0 to 12; b is an integer of 0 or 1; g is an integer of 0 or1; Ab is a moiety comprising at least one antigen binding site; and n isthe ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] to the moietycomprising at least one antigen binding site and is in the range from 1to
 20. 12. A drug conjugate according to claim 11, or a pharmaceuticallyacceptable salt, ester, solvate, tautomer or stereoisomer thereof,wherein D is a drug moiety selected from formulas (IHa) and (IHb):

where the wavy lines, R₁, R₂, R₃, Y, and Z are as defined for formula(IH).
 13. The drug conjugate according to any one of claims 1 to 4, 11or 12, wherein Y is —NH—.
 14. The drug conjugate according to any one ofclaims 1 to 4, 11 or 12, wherein Y is —O—.
 15. The drug conjugateaccording to any one of claims 1 to 4, or 11 to 14, wherein R₁ is —OH.16. The drug conjugate according to any one of claims 1 to 4, or 11 to14, wherein R₁ is —CN.
 17. The drug conjugate according to any one ofclaims 1 to 4, or 11 to 16, wherein R₂ is a —C(═O)R_(a) group whereR_(a) is substituted or unsubstituted C₁-C₆ alkyl; preferably whereinR_(a) is selected from substituted or unsubstituted methyl, substitutedor unsubstituted ethyl, substituted or unsubstituted n-propyl,substituted or unsubstituted isopropyl, substituted or unsubstitutedn-butyl, substituted or unsubstituted isobutyl, substituted orunsubstituted sec-butyl and substituted or unsubstituted tert-butyl. 18.The drug conjugate according to claim 17 wherein R₂ is acetyl.
 19. Thedrug conjugate according to any one of claims 1 to 4, or 11 to 18,wherein R₃ is hydrogen or —OR_(b) wherein R_(b) is substituted orunsubstituted C₁-C₆ alkyl; preferably wherein R_(b) is selected fromsubstituted or unsubstituted methyl, substituted or unsubstituted ethyl,substituted or unsubstituted n-propyl, substituted or unsubstitutedisopropyl, substituted or unsubstituted n-butyl, substituted orunsubstituted isobutyl, substituted or unsubstituted sec-butyl andsubstituted or unsubstituted tert-butyl.
 20. The drug conjugateaccording to claim 19 wherein R₃ is hydrogen.
 21. The drug conjugateaccording to claim 19 wherein R₃ is —OR_(b) wherein R_(b) is substitutedor unsubstituted C₁-C₆ alkyl; preferably wherein R_(b) is selected fromsubstituted or unsubstituted methyl, substituted or unsubstituted ethyl,substituted or unsubstituted n-propyl, substituted or unsubstitutedisopropyl, substituted or unsubstituted n-butyl, substituted orunsubstituted isobutyl, substituted or unsubstituted sec-butyl andsubstituted or unsubstituted tert-butyl.
 22. The drug conjugateaccording to claim 21 wherein R₃ is methoxy.
 23. The drug conjugateaccording to any one of claims 1 to 22, wherein the salt is selectedfrom hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate,phosphate, acetate, trifluoroacetate, maleate, fumarate, citrate,oxalate, succinate, tartrate, malate, mandelate, methanesulfonate,p-toluenesulfonate, sodium, potassium, calcium, ammonium,ethylenediamine, ethanolamine, N,N-dialkylenethanolamine,triethanolamine and basic aminoacids.
 24. A drug conjugate according toany one of the preceding claims, wherein L is a linker group selectedfrom the group consisting of:

wherein the wavy lines indicate the point of covalent attachments to anAb (the wavy line to the right) and to (T)_(g) if any, or (AA)_(w) ifany, or (X)_(b) if any, or D (the wavy line to the left); R₁₉ isselected from —C₁-C₁₂ alkylene-, —C₃-C₈ carbocyclo, —O—C₁-C₁₂ alkylene),—C₆-C₁₈ arylene in one or more rings which may optionally be substitutedwith one or more substituents R_(x), —C₁-C₁₂ alkylene-C₆-C₁₈ arylene-wherein the arylene group is in one or more rings which may optionallybe substituted with one or more substituents R_(x), —C₆-C₁₈arylene-C₁-C₁₂ alkylene-wherein the arylene group is in one or morerings which may optionally be substituted with one or more substituentsR_(x), —C₁-C₁₂ alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈ carbocyclo)-C₁-C₁₂alkylene-, —C₅-C₁₄ heterocyclo- wherein said heterocyclo group may be asaturated or unsaturated group having one or more rings and comprisingat least one oxygen, nitrogen or sulphur atom in said ring(s), saidgroup optionally being substituted with one or more substituents R_(x),—C₁-C₁₂ alkylene-(C₅-C₁₄ heterocyclo)-wherein said heterocyclo group maybe a saturated or unsaturated group having one or more rings andcomprising at least one oxygen, nitrogen or sulphur atom in saidring(s), said group optionally being substituted with one or moresubstituents R_(x), —(C₅-C₁₄ heterocyclo)-C₁-C₁₂ alkylene- wherein saidheterocyclo group may be a saturated or unsaturated group having one ormore rings and comprising at least one oxygen, nitrogen or sulphur atomin said ring(s), said group optionally being substituted with one ormore substituents R_(x), —(OCH₂CH₂)_(r)— and —CH₂—(OCH₂CH₂)_(r)—,wherein each of the above alkylene substituents whether alone orattached to another moiety the carbon chain may optionally besubstituted by one or more substituents R_(x); R₃₀ is a —C₁-C₆ alkylene-group; M is selected from the group consisting of —C₁-C₆ alkylene-,—C₁-C₆ alkylene-(C₃-C₈carbocyclo)-, —(CH₂CH₂O)_(s)—, —C₁-C₆alkylene-(C₃-C₈ carbocyclo)-CON(H or C₁-C₆ alkyl)-C₁-C₆ alkylene-,phenylene which may optionally be substituted with one or moresubstituents R_(x), phenylene-C₁-C₆ alkylene- wherein the phenylenemoiety may optionally be substituted with one or more substituents R_(x)and —C₁-C₆ alkylene-CON(H or C₁-C₆alkyl)C₁-C₆ alkylene-; Q is selectedfrom the group consisting of —N(H or C₁-C₆ alkyl)phenylene- and —N(H orC₁-C₆ alkyl)-(CH₂)_(s); r is an integer ranging from 1 to 10; and s isan integer ranging from 1 to
 10. 25. A drug conjugate according to anyof claims 1 to 24, wherein L is a linker group selected from the groupconsisting of:

wherein: the wavy lines indicate the point of covalent attachments to anAb (the wavy line to the right) and to (T)_(g) if any, or (AA)_(w) ifany, or to (X)_(b) (the wavy line to the left); R₁₉ is selected from—C₁-C₁₂ alkylene-, —O—(C₁-C₁₂ alkylene), —C₆-C₁₂ arylene in one or morerings which may optionally be substituted with one or more substituentsR_(x), —C₁-C₁₂ alkylene-C₆-C₁₂ arylene- wherein the arylene group is inone or more rings which may optionally be substituted with one or moresubstituents R_(x), —C₆-C₁₂ arylene-C₁-C₁₂ alkylene- wherein the arylenegroup is in one or more rings which may optionally be substituted withone or more substituents R_(x), —C₅-C₁₂ heterocyclo- wherein saidheterocyclo group may be a saturated or unsaturated group having one ormore rings and comprising at least one oxygen, nitrogen or sulphur atomin said ring(s), said group optionally being substituted with one ormore substituents R_(x), —C₁-C₁₂ alkylene-(C₅-C₁₂ heterocyclo)- whereinsaid heterocyclo group may be a saturated or unsaturated group havingone or more rings and comprising at least one oxygen, nitrogen orsulphur atom in said ring(s), said group optionally being substitutedwith one or more substituents R_(x), —(C₅-C₁₂ heterocyclo)-C₁-C₁₂alkylene- wherein said heterocyclo group may be a saturated orunsaturated group having one or more rings and comprising at least oneoxygen, nitrogen or sulphur atom in said ring(s), said group optionallybeing substituted with one or more substituents R_(x), —(OCH₂CH₂)_(r)—and —CH₂—(OCH₂CH₂)_(r)—, wherein each of the above alkylene substituentswhether alone or attached to another moiety the carbon chain mayoptionally be substituted by one or more substituents R_(x); R₃₀ is a—C₁-C₆ alkylene- group; M is selected from the group consisting of—C₁-C₆ alkylene-, —C₁-C₆ alkylene-(C₃-C₈carbocyclo)- and phenylene whichmay optionally be substituted with one or more substituents R_(x); and ris an integer ranging from 1-6.
 26. A drug conjugate according to anyone of claims 1 to 25, selected from the formulas (IV), (V) and (VI):

wherein: X and T are extending groups that may be the same or different;each AA is independently an amino acid unit; w is an integer rangingfrom 0 to 12; b is an integer of 0 or 1; g is an integer of 0 or 1; D isa drug moiety; Ab is a moiety comprising at least one antigen bindingsite; n is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]wherein L is as defined in formula (IV), (V) or (VI) to the moietycomprising at least one antigen binding site and is in the range from 1to 20; R₁₉ is selected from —C₁-C₈ alkylene-, —O—(C₁-C₈ alkylene),—C₁-C₈ alkylene-C₆-C₁₂ arylene-wherein the arylene group is in one ormore rings which may optionally be substituted with one or moresubstituents R_(x), and —C₆-C₁₂ arylene-C₁-C₈ alkylene- wherein thearylene group is in one or more rings which may optionally besubstituted with one or more substituents R_(x), wherein each of theabove alkylene substituents whether alone or attached to another moietythe carbon chain may optionally be substituted by one or moresubstituents R_(x); R₃₀ is a —C₂-C₄ alkylene- group; and M is selectedfrom the group consisting of —C₁-C₃ alkylene- and —C₁-C₃ alkylene-(C₅-C₇carbocyclo)-.
 27. A drug conjugate according to claim 26 selected fromthe formulas (IV), (V) and (VI):

wherein: X and T are extending groups that may be the same or different;each AA is independently an amino acid unit; w is an integer rangingfrom 0 to 12; b is an integer of 0 or 1; g is an integer of 0 or 1; D isa drug moiety; Ab is a moiety comprising at least one antigen bindingsite; n is the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-]wherein L is as defined in (IV), (V) or (VI) to the moiety comprising atleast one antigen binding site and is in the range from 1 to 20; R₁₉ isselected from —C₁-C₆ alkylene-, phenylene-C₁-C₆ alkylene- wherein thephenylene group may optionally be substituted with one or moresubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, halogen atoms, nitro groups and cyano groups, wherein each of theabove alkylene substituents whether alone or attached to another moietyin the carbon chain may optionally be substituted by one or moresubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, aryl groups having from 6 to 12 carbon atoms, halogen atoms,nitro groups and cyano groups, and preferably R₁₉ is a —C₁-C₆ alkylenegroup; R₃₀ is a —C₂-C₄ alkylene- group; and M is —C₁-C₃alkylene-(C₅-C₇carbocyclo)-.
 28. A drug conjugate according to any oneof claims 1 to 27, wherein (AA)_(w) is of formula (II):

wherein the wavy lines indicate the point of covalent attachments to(X)_(b) if any, or to the drug moiety (the wavy line to the left) and to(T)_(g) if any, or to the linker (the wavy line to the right); and R₂₁is, at each occurrence, selected from the group consisting of hydrogen,methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH₂OH,—CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH,—(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂, —(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO,—(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂, —(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO,—(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂, CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-,3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

and w is an integer ranging from 0 to
 12. 29. A drug conjugate accordingto claim 28, wherein (AA)_(w) is of formula (II) wherein: R₂₁ isselected, at each occurrence, from the group consisting of hydrogen,methyl, isopropyl, sec-butyl, benzyl, indolylmethyl, —(CH₂)₃NHCONH₂,—(CH₂)₄NH₂, —(CH₂)₃NHC(═NH)NH₂ and —(CH₂)₄NHC(═NH)NH₂; and w is aninteger ranging from 0 to
 6. 30. A drug conjugate according to any oneof claims 1 to 28, wherein w is 0 or 2, and where w is 2, then (AA)_(w)is of formula (III):

wherein: the wavy lines indicate the point of covalent attachments to(X)_(b) if any, or to the drug moiety (the wavy line to the left) and to(T)_(g) if any, or to the linker (the wavy line to the right); R₂₂ isselected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl;and R₂₃ is selected from methyl, —(CH₂)₄NH₂, —(CH₂)₃NHCONH₂ and—(CH₂)₃NHC(═NH)NH₂.
 31. A drug conjugate according to any one of claims1 to 30, wherein X is an extending group selected from: where D iscovalently attached via an amine group (for example where Z is —NH—):—COO—(C₁-C₆ alkylene)NH—; —COO—CH₂-(phenylene which may optionally besubstituted with one or more substituents R_(x))—NH—; —COO—(C₁-C₆alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withone or more substituents R_(x))—NH—; —COCH₂NH—COCH₂—NH—; —COCH₂NH—;—COO—(C₁-C₆ alkylene)S—; —COO—(C₁-C₆ alkylene)NHCO(C₁-C₆ alkylene)S—;and where D is covalently attached via an hydroxy group (for examplewhere Z is —O—): —CONH—(C₁-C₆ alkylene)NH—; —COO—CH₂-(phenylene whichmay optionally be substituted with one or more substituents R_(x))—NH—;—CONH—(C₁-C₆ alkylene)NH—COO—CH₂-(phenylene which may optionally besubstituted with one or more substituents R_(x))—NH—;—COCH₂NH—COCH₂—NH—; —COCH₂NH—; —CONH—(C₁-C₆ alkylene)S—; —CONH—(C₁-C₆alkylene)NHCO(C₁-C₆ alkylene)S—; and b is 0 or 1, preferably
 1. 32. Adrug conjugate according to any one of claims 1 to 31, wherein X is anextending group selected from the group consisting of: where D iscovalently attached via an amine group (for example where Z is —NH—):—COO—(C₂-C₄ alkylene)NH—; —COO—CH₂-phenylene-NH—, wherein said phenylenegroup may optionally be substituted with from one to four substituentsR_(x) selected from the group consisting of alkyl groups having from 1to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,halogen atoms, nitro groups and cyano groups; —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—; —COCH₂NH—COCH₂—NH—; —COO—(C₂-C₄ alkylene)S—; —COO—(C₂-C₄alkylene)NHCO(C₁-C₃ alkylene)S—; or where D is covalently attached viaan hydroxy group (for example where Z is —O—): —CONH—(C₂-C₄alkylene)NH—; —COO—CH₂-phenylene-NH—, wherein said phenylene group mayoptionally be substituted with from one to four substituents R_(x)selected from the group consisting of alkyl groups having from 1 to 6carbon atoms, alkoxy groups having from 1 to 6 carbon atoms, halogenatoms, nitro groups and cyano groups; —CONH—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—; —COCH₂NH—COCH₂—NH—; —CONH—(C₂-C₄ alkylene)S—; —CONH—(C₂-C₄alkylene)NHCO(C₁-C₃ alkylene)S—; and b is 0 or 1, preferably
 1. 33. Adrug conjugate according to claim 32, wherein X is an extending groupselected from the group consisting of: where D is covalently attachedvia an amine group (for example where Z is —NH—): —COO-CH₂-phenylene-NH——COO(CH₂)₃NHCOOCH₂-phenylene-NH—; —COO(CH₂)₃NH—; —COO(CH₂)₃—S—;—COO(CH₂)₃NHCO(CH₂)₂S—; or where D is covalently attached via an hydroxygroup (for example where Z is —O—): —COO—CH₂-phenylene-NH——CONH(CH₂)₃NHCOOCH₂-phenylene-NH—; —CONH(CH₂)₃NH—; —CONH(CH₂)₃—S—;—CONH(CH₂)₃NHCO(CH₂)₂S—; and b is 0 or 1, preferably
 1. 34. A drugconjugate according to any one of claims 1 to 33, wherein T is anextending group selected from the group consisting of —CO—(C₁-C₆alkylene)-NH—, —CO—(C₁-C₆ alkylene)-[O—(C₂-C₆alkylene)]_(j)-NH—,—COO—(C₁-C₆ alkylene)-[O—(C₂-C₆ alkylene)]_(j)-NH—; where j is aninteger from 1 to 25, and g is 0 or
 1. 35. A drug conjugate according toclaim 34, wherein T is an extending group selected from the groupconsisting of —CO—(C₁-C₄ alkylene)NH—, —CO—(C₁-C₄ alkylene)-[O—(C₂-C₄alkylene)]_(j)-NH—, —COO—(C₁-C₄ alkylene)-[O—(C₂-C₄alkylene)]_(j)-NH—,where j is an integer from 1 to 10; and g is 0 or 1.36. A drug conjugate according to claim 35, wherein T is an extendinggroup selected from the group consisting of —CO—(C₁-C₄ alkylene)NH—,—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—; where j is an integer from 1 to5; and g is 0 or 1
 37. A drug conjugate according to any one of claims11 to 36, wherein D is a drug moiety of formula (IHa) or formula (IHb)or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof, wherein: R₁ is CN or OH; R₂ is C(═O)R_(a), whereinR_(a) is selected from hydrogen and substituted or unsubstituted C₁-C₆alkyl, wherein the optional substituents are one or more substituentsR_(x); R₃ is hydrogen or a —OR_(b) group wherein R_(b) is a substitutedor unsubstituted C₁-C₆ alkyl group, wherein the optional substituentsare one or more substituents R_(x), Y is —NH— or —O—; and Z is —NH— or—O—.
 38. A drug conjugate according to claim 37, wherein D is a drugmoiety of formula (IHa) or formula (IHb) or a pharmaceuticallyacceptable salt, ester, solvate, tautomer or stereoisomer thereof,wherein: R₁ is CN or OH; R₂ is acetyl; R₃ is hydrogen or methoxy, morepreferably hydrogen; Y is —NH— or —O—; and Z is —NH— or —O—.
 39. A drugconjugate according to claim 37 or claim 38 wherein D is a drug moietyof formula (IHa) or formula (IHb), or a pharmaceutically acceptablesalt, ester, solvate, tautomer or stereoisomer thereof wherein: R₁ isCN; R₂ is acetyl: R₃ is hydrogen; Y is —NH— or —O—; and Z is —NH—.
 40. Adrug conjugate according to any one of claims 1, 3, 4, 11, or 13 to 39,wherein D is selected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof, wherein the wavy line indicates the point ofcovalent attachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g)if any, or to (L).
 41. A drug conjugate according to any one of claims 1to 4 or 11 to 40, wherein D is

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof, wherein the wavy line indicates the point ofcovalent attachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g)if any, or to (L).
 42. A drug conjugate according to any one of claims 1to 41, wherein the moiety Ab comprising at least one antigen bindingsite is an antigen-binding peptide.
 43. A drug conjugate according toclaim 42, wherein the moiety Ab comprising at least one antigen bindingsite is an antibody, a single domain antibody or an antigen-bindingfragment thereof.
 44. A drug conjugate according to claim 42 or 43,wherein the moiety Ab comprising at least one antigen binding site is amonoclonal antibody, polyclonal antibody or bispecific antibody andwherein the antibody or an antigen-binding fragment thereof is derivedfrom any species, preferably a human, mouse or rabbit.
 45. A drugconjugate according to claim 43 or 44, wherein the antibody orantigen-binding fragment thereof is selected from the group consistingof a human antibody, an antigen-binding fragment of a human antibody, ahumanized antibody, an antigen-binding fragment of a humanized antibody,a chimeric antibody, an antigen-binding fragment of a chimeric antibody,a glycosylated antibody and a glycosylated antigen binding fragment. 46.A drug conjugate according to any one of claims 43 to 45, wherein theantibody or antigen-binding fragment thereof is an antigen-bindingfragment selected from the group consisting of an Fab fragment, an Fab′fragment, an F(ab′)₂ fragment and an Fv fragment.
 47. A drug conjugateaccording to any one of claims 43 to 46, wherein the antibody orantigen-binding fragment thereof is a monoclonal antibody whichimmunospecifically binds to cancer cell antigens, viral antigens,antigens of cells that produce autoimmune antibodies associated withautoimmune disease, microbial antigens, and preferably a monoclonalantibody which immunospecifically binds to cancer cell antigens.
 48. Adrug conjugate according to any one of claims 1 to 47, wherein themoiety Ab comprising at least one antigen binding site is an antibodyselected from the group consisting of Abciximab, Alemtuzumab, Anetumab,Atezolizumab, Avelumab, Basiliximab, Bevacizumab, Blinatomumab,Brentuximab, Catumaxomab, Cetuximab, Coltuximab, Daclizumab,Daratumumab, Denintuzumab, Denosumab, Depatuxizumab, Dinutuximab,Durvalumab, Elotuzumab, Enfortumab, Glembatumumab, Gemtuzumab,Ibritumomab, Indatuximab, Indusatumab, Inotuzumab, Ipilimumab,Labetuzumab, Ladiratuzumab, Laprituximab, Lifastuzumab, Lorvotuzumab,Milatuzumab, Mirvetuximab, Naratuximab, Necitumumab, Nimotuzumab,Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Omalizumab,Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab, Pinatuzumab,Polatuzumab, Ramucirumab, Rovalpituzumab, Sacituzumab, Siltuximab,Sirtratumab, Sofituzumab, Vadastuximab, Vorsetuzumab, Trastuzumab, ananti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and ananti-CD 30 antibody, or an antigen-binding fragment or animmunologically active portion thereof.
 49. A drug conjugate accordingto claim 48, wherein the moiety Ab comprising at least one antigenbinding site is an antibody selected from the group consisting ofAbciximab, Alemtuzumab, Anetumab, Atezolizumab, Avelumab, Basiliximab,Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab,Daclizumab, Daratumumab, Denintuzumab, Denosumab, Depatuxizumab,Dinutuximab, Durvalumab, Elotuzumab, Enfortumab, Glembatumumab,Gemtuzumab, Ibritumomab, Indatuximab, Indusatumab, Inotuzumab,Ipilimumab, Labetuzumab, Ladiratuzumab, Laprituximab, Mirvetuximab,Naratuximab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab,Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab,Pembrolizumab, Pertuzumab, Polatuzumab, Ramucirumab, Rovalpituzumab,Sacituzumab, Siltuximab, Sirtratumab, Vadastuximab, Vorsetuzumab,Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13antibody and an anti-CD 30 antibody, or an antigen-binding fragment oran immunologically active portion thereof.
 50. A drug conjugateaccording to claim 48, wherein the moiety Ab comprising at least oneantigen binding site is an antibody selected from the group consistingof Abciximab, Alemtuzumab, Atezolizumab, Avelumab, Basiliximab,Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab,Daclizumab, Daratumumab, Denosumab, Dinutuximab, Durvalumab, Elotuzumab,Gemtuzumab, Ibritumomab, Inotuzumab, Ipilimumab, Labetuzumab,Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab,Olaratumab, Omalizumab, Palivizumab, Panitumumab, Pembrolizumab,Pertuzumab, Ramucirumab, Rovalpituzumab, Siltuximab, Trastuzumab, ananti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and ananti-CD 30 antibody, or an antigen-binding fragment or animmunologically active portion thereof, more preferably Brentuximab,Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30antibody, or an antigen-binding fragment or an immunologicallly activeportion thereof, preferably Trastuzumab and anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof,more preferably Trastuzumab or an antigen-binding fragment or animmunologically active portion thereof.
 51. A drug conjugate accordingto claim 11 or claim 12, wherein: L is a linker group selected from thegroup consisting of:

wherein: the wavy lines indicate the point of covalent attachments to anAb (the wavy line to the right) and to (T)_(g) if any, or (AA)_(w) ifany, or to (X)_(b) if any, or to D (the wavy line to the left); R₁₉ isselected from —C₁-C₁₂ alkylene-, —O—(C₁-C₁₂ alkylene), —C₆-C₁₂ arylenein one or more rings which may optionally be substituted with one ormore substituents R_(x), —C₁-C₁₂ alkylene-C₆-C₁₂ arylene- wherein thearylene group is in one or more rings which may optionally besubstituted with one or more substituents R_(x), —C₆-C₁₂ arylene-C₁-C₁₂alkylene- wherein the arylene group is in one or more rings which mayoptionally be substituted with one or more substituents R_(x), —C₅-C₁₂heterocyclo- wherein said heterocyclo group may be a saturated orunsaturated group having one or more rings and comprising at least oneoxygen, nitrogen or sulphur atom in said ring(s), said group optionallybeing substituted with one or more substituents R_(x), —C₁-C₁₂alkylene-(C₅-C₁₂ heterocyclo)-wherein said heterocyclo group may be asaturated or unsaturated group having one or more rings and comprisingat least one oxygen, nitrogen or sulphur atom in said ring(s), saidgroup optionally being substituted with one or more substituents R_(x),—(C₅-C₁₂ heterocyclo)-C₁-C₁₂ alkylene- wherein said heterocyclo groupmay be a saturated or unsaturated group having one or more rings andcomprising at least one oxygen, nitrogen or sulphur atom in saidring(s), said group optionally being substituted with one or moresubstituents R_(x), —(OCH₂CH₂)_(r)— and —CH₂—(OCH₂CH₂)_(r)—, whereineach of the above alkylene substituents whether alone or attached toanother moiety the carbon chain may optionally be substituted by one ormore substituents R_(x); R₃₀ is a —C₁-C₆ alkylene- group; M is selectedfrom the group consisting of —C₁-C₆ alkylene-, —C₁-C₆alkylene-(C₃-C₈carbocyclo)- and phenylene which may optionally besubstituted with one or more substituents R_(x); r is an integer rangingfrom 1-6; (AA)_(w) is of formula (II):

wherein the wavy lines indicate the point of covalent attachments to(X)_(b) if any, or to the drug moiety (the wavy line to the left) and to(T)_(g) if any, or to the linker (the wavy line to the right); R₂₁ is,at each occurrence, selected from the group consisting of hydrogen,methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, —CH₂OH,—CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH, —CH₂CH₂CONH₂, —CH₂CH₂COOH,—(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂, —(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO,—(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂, —(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO,—(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂, CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-,3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

w is an integer ranging from 0 to 12; wherein X is an extending groupselected from where Z is —NH—: —COO—(C₁-C₆ alkylene)NH—,—COO—CH₂-(phenylene which may optionally be substituted with one or moresubstituents R_(x))—NH—, —COO—(C₁-C₆ alkylene)NH—COO—CH₂-(phenylenewhich may optionally be substituted with one or more substituentsR_(x))—NH—, —COCH₂NH—COCH₂—NH—, —COCH₂—NH—, —COO—(C₁-C₆ alkylene)S—,—COO—(C₁-C₈ alkylene)NHCO(C₁-C₆ alkylene)S—; or where Z is —O-:—CONH—(C₁-C₆ alkylene)NH—, —COO—CH₂-(phenylene which may optionally besubstituted with one or more substituents R_(x))—NH—, —CONH—(C₁-C₆alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withone or more substituents R_(x))—NH—, —COCH₂NH—COCH₂—NH—, —COCH₂NH—,—CONH—(C₁-C₆ alkylene)S—, —CONH—(C₁-C₆ alkylene)NHCO(C₁-C₆ alkylene)S—;b is 0 or 1, preferably 1; wherein T is an extending group selected from—CO—(C₁-C₆ alkylene)-NH—, —CO—(C₁-C₆ alkylene)-[O—(C₂-C₆alkylene)]_(j)-NH—, and —COO—(C₁-C₆ alkylene)-[O—(C₂-C₆alkylene)]_(j)-NH—, where j is an integer from 1 to 25; g is 0 or 1; Dis a drug moiety of formula (IH), formula (IHa) or formula (IHb), or apharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof wherein: R₁ CN or OH; R₂ is C(═O)R_(a), whereinR_(a) is selected from hydrogen and substituted or unsubstituted C₁-C₆alkyl, wherein the optional substituents are one or more substituentsR_(x); R₃ is hydrogen or a —OR_(b) group wherein R_(b) is a substitutedor unsubstituted C₁-C₆ alkyl group, wherein the optional substituentsare one or more substituents R_(x); Y is —NH— or —O—; Z is —NH— or —O—;the moiety Ab comprising at least one antigen binding site is anantibody or an antigen-binding fragment thereof and it is selected fromthe group consisting of a human antibody, an antigen-binding fragment ofa human antibody, a humanized antibody, an antigen-binding fragment of ahumanized antibody, a chimeric antibody, an antigen-binding fragment ofa chimeric antibody, a glycosylated antibody and a glycosylated antigenbinding fragment; and n is the ratio of the group[D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] to the moiety Ab comprising at leastone antigen binding site and is in the range from 1 to
 12. 52. A drugconjugate according to claim 11 or claim 12, selected from the formulas(IV), (V), and (VI):

wherein: R₁₉ is selected from —C₁-C₈ alkylene-, —O—(C₁-C₈ alkylene),—C₁-C₈ alkylene-C₆-C₁₂ arylene-wherein the arylene group is in one ormore rings which may optionally be substituted with one or moresubstituents R_(x) and —C₆-C₁₂ arylene-C₁-C₈ alkylene- wherein thearylene group is in one or more rings which may optionally besubstituted with one or more substituents R_(x), wherein each of theabove alkylene substituents whether alone or attached to another moietythe carbon chain may optionally be substituted by one or moresubstituents R_(x); R₃₀ is a —C₂-C₄ alkylene- group; M is selected fromthe group consisting of —C₁-C₃ alkylene- and —C₁-C₃ alkylene-(C₅-C₇carbocyclo)-; (AA)_(w) is of formula (II)

wherein: the wavy lines indicate the point of covalent attachments to(X)_(b) if any, or to the drug moiety (the wavy line to the left) and to(T)_(g) if any, or to the linker (the wavy line to the right); R₂₁ is,at each occurrence, selected from the group consisting of hydrogen,methyl, isopropyl, sec-butyl, benzyl, indolylmethyl, —(CH₂)₃NHCONH₂,—(CH₂)₄NH₂, —(CH₂)₃NHC(═NH)NH₂ and —(CH₂)₄NHC(═NH)NH₂; w is an integerfrom 0 to 6; X is an extending group selected from the group consistingof where Z is —NH—: —COO—(C₂-C₄ alkylene)NH—, —COO-CH₂-phenylene-NH—,wherein said phenylene group may optionally be substituted with from oneto four substituents R_(x) selected from the group consisting of alkylgroups having from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6carbon atoms, halogen atoms, nitro groups and cyano groups, —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —COO—(C₂-C₄ alkylene)S—, and—COO—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—; or where Z is —O-:—CONH—(C₂-C₄ alkylene)NH—, —COO-CH₂-phenylene-NH—, wherein saidphenylene group may optionally be substituted with from one to foursubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, halogen atoms, nitro groups and cyano groups, —CONH—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —CONH—(C₂-C₄ alkylene)S—, and—CONH—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—; b is 0 or 1, preferably 1;wherein T is an extending group selected from —CO—(C₁-C₄ alkylene)-NH—,—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to10; g is 0 or 1; D is a drug moiety of formula (IH), formula (IHa) orformula (IHb), or a pharmaceutically acceptable salt, ester, solvate,tautomer or stereoisomer thereof wherein: R₁ is CN or OH; R₂ is acetyl;R₃ is hydrogen or methoxy, more preferably hydrogen; Y is —NH— or —O—; Zis —NH— or —O—; the moiety Ab comprising at least one antigen bindingsite is an antibody or an antigen-binding fragment thereof, wherein theantibody or antigen-binding fragment is a monoclonal antibody whichimmunospecifically binds to cancer cell antigens, viral antigens,antigens of cells that produce autoimmune antibodies associated withautoimmune disease, microbial antigens, and preferably a monoclonalantibody which immunospecifically binds to cancer cell antigens; and nis the ratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein L isas defined in formulas (IV), (V) or (VI) to the moiety Ab comprising atleast one antigen binding site and is in the range from 3 to
 8. 53. Adrug conjugate according to claim 11 or claim 12, selected from theformulas (IV), (V) and (VI):

wherein: R₁₉ is selected from —C₁-C₆ alkylene-, -phenylene-C₁-C₆alkylene- wherein the phenylene group may optionally be substituted withone or more substituents R_(x) selected from the group consisting ofalkyl groups having from 1 to 6 carbon atoms, alkoxy groups having from1 to 6 carbon atoms, halogen atoms, nitro groups and cyano groups,wherein each of the above alkylene substituents whether alone orattached to another moiety in the carbon chain may optionally besubstituted by one or more substituents R_(x) selected from the groupconsisting of alkyl groups having from 1 to 6 carbon atoms, alkoxygroups having from 1 to 6 carbon atoms, aryl groups having from 6 to 12carbon atoms, halogen atoms, nitro groups and cyano groups, andpreferably R₁₉ is a C₁-C₆ alkylene group; R₃₀ is a —C₂-C₄ alkylene-group; M is —C₁-C₃ alkylene-(C₅-C₇carbocyclo)-; w is 0 or 2, and where wis 2, then (AA)_(w) is of formula (III):

wherein the wavy lines indicate the point of covalent attachments to(X)_(b) if any, or to the drug moiety (the wavy line to the left) and to(T)_(g) if any, or to the linker (the wavy line to the right); R₂₂ isselected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl;R₂₃ is selected from methyl, —(CH₂)₄NH₂, —(CH₂)₃NHCONH₂ and—(CH₂)₃NHC(═NH)NH₂; X is an extending group selected from the groupconsisting of —COO—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH—, whereinsaid phenylene group may optionally be substituted with from one to foursubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, halogen atoms, nitro groups and cyano groups, —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups or cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —COO—(C₂-C₄ alkylene)S—, and—COO—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—; b is 0 or 1, preferably 1;wherein T is an extending group selected from —CO—(C₁-C₄ alkylene)-NH—,—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to5; g is 0 or 1; D is a drug moiety of formula (IHa) or formula (IHb), ora pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof wherein: R₁ is CN; R₂ is acetyl: R₃ is hydrogen; Yis —NH— or —O—; Z is —NH—; the moiety Ab comprising at least one antigenbinding site is a monoclonal antibody selected from the group consistingof Abciximab, Alemtuzumab, Anetumab, Atezolizumab, Avelumab,Basiliximab, Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab,Cetuximab, Coltuximab, Daclizumab, Daratumumab, Denintuzumab, Denosumab,Depatuxizumab, Dinutuximab, Durvalumab, Elotuzumab, Enfortumab,Glembatumumab, Gemtuzumab, Ibritumomab, Indatuximab, Indusatumab,Inotuzumab, Ipilimumab, Labetuzumab, Ladiratuzumab, Laprituximab,Lifastuzumab, Lorvotuzumab, Milatuzumab, Mirvetuximab, Naratuximab,Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab,Olaratumab, Omalizumab, Palivizumab, Panitumumab, Pembrolizumab,Pertuzumab, Pinatuzumab, Polatuzumab, Ramucirumab, Rovalpituzumab,Sacituzumab, Siltuximab, Sirtratumab, Sofituzumab, Vadastuximab,Vorsetuzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody,an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-bindingfragment or an immunologically active portion thereof; preferablyAbciximab, Alemtuzumab, Atezolizumab, Avelumab, Basiliximab,Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab,Daclizumab, Daratumumab, Denosumab, Dinutuximab, Durvalumab, Elotuzumab,Gemtuzumab, Ibritumomab, Inotuzumab, Ipilimumab, Labetuzumab,Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab,Olaratumab, Omalizumab, Palivizumab, Panitumumab, Pembrolizumab,Pertuzumab, Ramucirumab, Rovalpituzumab, Siltuximab, Trastuzumab, ananti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and ananti-CD 30 antibody, or an antigen-binding fragment or animmunologically active portion thereof, more preferably Brentuximab,Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30antibody, or an antigen-binding fragment or an immunologically activeportion thereof, preferably Trastuzumab and anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof,more preferably Trastuzumab or an antigen-binding fragment or animmunologicallly active portion thereof; and n is the ratio of the group[D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein L is as defined in formulas(IV), (V) or (VI) to the moiety Ab comprising at least one antigenbinding site and is in the range from 3 to
 5. 54. A drug conjugateaccording to claim 53, the moiety Ab comprising at least one antigenbinding site is an antibody selected from the group consisting ofAbciximab, Alemtuzumab, Anetumab, Atezolizumab, Avelumab, Basiliximab,Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab,Daclizumab, Daratumumab, Denintuzumab, Denosumab, Depatuxizumab,Dinutuximab, Durvalumab, Elotuzumab, Enfortumab, Glembatumumab,Gemtuzumab, Ibritumomab, Indatuximab, Indusatumab, Inotuzumab,Ipilimumab, Labetuzumab, Ladiratuzumab, Laprituximab, Mirvetuximab,Naratuximab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab,Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab,Pembrolizumab, Pertuzumab, Polatuzumab, Ramucirumab, Rovalpituzumab,Sacituzumab, Siltuximab, Sirtratumab, Vadastuximab, Vorsetuzumab,Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13antibody and an anti-CD 30 antibody, or an antigen-binding fragment oran immunologically active portion thereof.
 55. A drug conjugateaccording to claim 53, wherein the moiety Ab comprising at least oneantigen binding site is an antibody selected from the group consistingof Abciximab, Alemtuzumab, Atezolizumab, Avelumab, Basiliximab,Bevacizumab, Blinatomumab, Brentuximab, Catumaxomab, Cetuximab,Daclizumab, Daratumumab, Denosumab, Dinutuximab, Durvalumab, Elotuzumab,Gemtuzumab, Ibritumomab, Inotuzumab, Ipilimumab, Labetuzumab,Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab,Olaratumab, Omalizumab, Palivizumab, Panitumumab, Pembrolizumab,Pertuzumab, Ramucirumab, Rovalpituzumab, Siltuximab, Trastuzumab, ananti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and ananti-CD 30 antibody, or an antigen-binding fragment or animmunologically active portion thereof, more preferably Brentuximab,Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, an anti-CD4antibody, an anti-CD5 antibody, an anti-CD13 antibody and an anti-CD 30antibody, or an antigen-binding fragment or an immunologically activeportion thereof, preferably Trastuzumab and anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof,more preferably Trastuzumab or an antigen-binding fragment or animmunologically active portion thereof.
 56. A drug conjugate accordingto claim 11, selected from the formulas (IV), (V) and (VI):

wherein: R₁₉ is —C₂-C₆ alkylene-; R₃₀ is a —C₂-C₄ alkylene-; M is —C₁-C₃alkylene-(C₅-C₇carbocyclo)-; w is 0 or 2, and where w is 2, then(AA)_(w) is of formula (III):

wherein R₂₂ is isopropyl, R₂₃ is selected from methyl and—(CH₂)₃NHCONH₂, wherein the wavy lines indicate the point of covalentattachments to (X)_(b) if any, or to the drug moiety (the wavy line tothe left) and to (T)_(g) if any, or to the linker (the wavy line to theright); X is an extending group selected from the group consisting of—COO—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH—, wherein said phenylenegroup may optionally be substituted with from one to four substituentsR_(x) selected from the group consisting of alkyl groups having from 1to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,halogen atoms, nitro groups and cyano groups, —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —COO—(C₂-C₄ alkylene)S—, and—COO—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S; b is 0 or 1, preferably 1;wherein T is an extending group selected from —CO—(C₁-C₄ alkylene)-NH—,—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to5; g is 0 or 1; D is a drug moiety selected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof; wherein the wavy lines indicate the point ofcovalent attachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g)if any, or to linker; the moiety Ab comprising at least one antigenbinding site is selected from Brentuximab, Gemtuzumab, Inozutumab,Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody,an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-bindingfragment or an immunologically active portion thereof; and n is theratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein L is asdefined in formulas (IV), (V) or (VI) to the moiety Ab comprising atleast one antigen binding site and is in the range from 3 to
 5. 57. Adrug conjugate according to claim 56, wherein the moiety Ab comprisingat least one antigen binding site is selected from Trastuzumab andanti-CD13 antibody or an antigen-binding fragment or an immunologicallyactive portion thereof.
 58. A drug conjugate according to claim 56,wherein the moiety Ab comprising at least one antigen binding site isTrastuzumab or an antigen-binding fragment or an immunologically activeportion thereof.
 59. A drug conjugate according to claim 11 or claim 12,selected from the formulas (IV), (V), and (VI):

wherein: R₁₉ is —C₂-C₆ alkylene-; R₃₀ is —C₂-C₄ alkylene-; M is —C₁-C₃alkylene-(C₅-C₇carbocyclo)-; w is 0 or 2, and where w is 2, then(AA)_(w) is of formula (III):

wherein R₂₂ is isopropyl, R₂₃ is selected from methyl and—(CH₂)₃NHCONH₂, and the wavy lines indicate the point of covalentattachments to (X)_(b) if any, or the drug moiety (the wavy line to theleft) and to (T)_(g) if any, or to the linker (the wavy line to theright); X is an extending group selected from the group consisting of—COO—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH—, wherein said phenylenegroup may optionally be substituted with from one to four substituentsR_(x) selected from the group consisting of alkyl groups having from 1to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,halogen atoms, nitro groups and cyano groups, —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —COO—(C₂-C₄ alkylene)S—, and—COO—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—; b is 0 or 1, preferably 1;wherein T is an extending group selected from —CO—(C₁-C₄ alkylene)-NH—,—CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to5; g is 0 or 1; D is a drug moiety selected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof; wherein the wavy lines indicate the point ofcovalent attachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g)if any, or to linker; the moiety Ab comprising at least one antigenbinding site is selected from Brentuximab, Gemtuzumab, Inozutumab,Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody,an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-bindingfragment or an immunologically active portion thereof; and n is theratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein L is asdefined in formulas (IV), (V) or (VI) to the moiety comprising at leastone antigen binding site and is in the range from 3 to
 5. 60. A drugconjugate according to claim 59, wherein the moiety Ab comprising atleast one antigen binding site is selected from Trastuzumab andanti-CD13 antibody or an antigen-binding fragment or an immunologicallyactive portion thereof.
 61. A drug conjugate according to claim 59,wherein the moiety Ab comprising at least one antigen binding site isTrastuzumab or an antigen-binding fragment or an immunologically activeportion thereof.
 62. A drug conjugate according to any one of claims 1to 4, or 11 to 49, of formula (IV):

wherein: R₁₉ is C₂-C₅ alkylene-; w is 0 or 2, and where w is 2, then(AA)_(w) is of formula (III):

wherein R₂₂ is isopropyl, R₂₃ is selected from methyl and—(CH₂)₃NHCONH₂, and the wavy lines indicate the point of covalentattachments to (X)_(b) if any, or to the drug moiety (the wavy line tothe left) and to (T)_(g) if any, or to the linker (the wavy line to theright); X is a —COOCH₂-phenylene-NH— group; b is 1; T is an extendinggroup of formula —CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]₄—NH—; g is 0or 1; or of formula (V)

wherein M is -methyl-cyclohexylene-; b is 1; w is 0; X is an extendinggroup selected from —(CH₂)₃S- and —(CH₂)₃NHCO(CH₂)₂S— g is 0; or offormula (VI)

wherein R₁₉ is —C₂-C₅ alkylene-; R₃₀ is —C₃ alkylene-; w is 0 or 2, andwhere w is 2, then (AA)_(w) is of formula (III):

wherein R₂₂ is isopropyl, R₂₃ is selected from methyl and—(CH₂)₃NHCONH₂, and the wavy lines indicate the point of covalentattachments to (X)_(b) if any, or to the drug moiety (the wavy line tothe left) and to (T)_(g) if any, or to the linker (the wavy line to theright); and X is a —COOCH₂-phenylene-NH group; b is 1; T is an extendinggroup of formula —CO—(C₁-C₄ alkylene)-[O—(C₂-C₄ alkylene)]₄—NH—; g is 0or 1; D is a drug moiety selected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof; wherein the wavy line indicates the point ofcovalent attachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g)if any, or to the linker; the moiety Ab comprising at least one antigenbinding site is is selected from Brentuximab, Gemtuzumab, Inozutumab,Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody,an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-bindingfragment or an immunologically active portion thereof; and n is theratio of the group [D-(X)_(b)-(AA)_(w)-(T)_(g)-(L)-] wherein L is asdefined in formula (IV) to the moiety comprising at least one antigenbinding site and is in the range from 3 to 5, and preferably
 4. 63. Adrug conjugate according to claim 62, wherein the moiety Ab comprisingat least one antigen binding site is selected from Trastuzumab andanti-CD13 antibody or an antigen-binding fragment or an immunologicallyactive portion thereof.
 64. A drug conjugate according to claim 62,wherein the moiety Ab comprising at least one antigen binding site isTrastuzumab or an antigen-binding fragment or an immunologically activeportion thereof.
 65. An antibody drug conjugate according to claim 1,selected from the group consisting of:

wherein n is from 2 to 6, more preferably 3, 4, or 5 and each

and

is independently selected from is independently selected fromBrentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab, ananti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and ananti-CD 30 antibody, or an antigen-binding fragment or animmunologically active portion thereof, and more preferably its isselected from Trastuzumab and anti-CD13 antibody or an antigen-bindingfragment or an immunologically active portion thereof, particularlyTrastuzumab or an antigen-binding fragment or an immunologically activeportion thereof.
 66. A drug conjugate according to claim 65, wherein themoiety Ab comprising at least one antigen binding site is selected fromTrastuzumab and anti-CD13 antibody or an antigen-binding fragment or animmunologically active portion thereof.
 67. A drug conjugate accordingto claim 65, wherein the moiety Ab comprising at least one antigenbinding site is selected from Trastuzumab or an antigen-binding fragmentor an immunologically active portion thereof.
 68. A drug conjugateaccording to claim 1, selected from the group consisting of:

wherein n is from 2 to 6, more preferably 3, 4, or 5 and each

and

is independently selected from Brentuximab, Gemtuzumab, Inozutumab,Rovalpituzumab, Trastuzumab, an anti-CD4 antibody, an anti-CD5 antibody,an anti-CD13 antibody and an anti-CD 30 antibody, or an antigen-bindingfragment or an immunologically active portion thereof, and morepreferably its is selected from Trastuzumab and anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof,particularly Trastuzumab or an antigen-binding fragment or animmunologically active portion thereof; or a drug conjugate of formula

wherein n is from 2 to 6, more preferably 3, 4, or 5 and

is an anti-CD13 antibody or an antigen-binding fragment or animmunologically active portion thereof.
 69. A drug conjugate accordingto claim 68, wherein the moiety Ab comprising at least one antigenbinding site is selected from Trastuzumab and anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereof.70. A drug conjugate according to claim 68, wherein the moiety Abcomprising at least one antigen binding site is selected fromTrastuzumab or an antigen-binding fragment or an immunologically activeportion thereof.
 71. An antibody drug conjugate according to any one ofclaims 1 to 70 in isolated or purified form.
 72. A compound of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-L₁, wherein: L₁ is a linker selected from thegroup of formulas consisting of:

wherein each of the the wavy lines indicates the point of covalentattachment to (T)_(g) if any, or (AA)_(w) if any, or to (X)_(b) if anyor to D; G is selected from halo, —O-mesyl and —O-tosyl; J is selectedfrom halo, hydroxy, —N-succinimidoxy, —O-(4-nitrophenyl),—O-pentafluorophenyl, —O-tetrafluorophenyl and —O—C(O)-OR₂₀; R₁₉ isselected from —C₁-C₁₂ alkylene-, —C₃-C₈ carbocyclo, —O—(C₁-C₁₂alkylene), —C₆-C₁₈ arylene in one or more rings which may optionally besubstituted with one or more substituents R_(x), —C₁-C₁₂ alkylene-C₆-C₁₈arylene- wherein the arylene group is in one or more rings which mayoptionally be substituted with one or more substituents R_(x), —C₆-C₁₈arylene-C₁-C₁₂ alkylene-wherein the arylene group is in one or morerings which may optionally be substituted with one or more substituentsR_(x), —C₁-C₁₂ alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈ carbocyclo)-C₁-C₁₂alkylene-, —C₅-C₁₄ heterocyclo- wherein said heterocyclo group may be asaturated or unsaturated group having one or more rings and comprisingat least one oxygen, nitrogen or sulphur atom in said ring(s), saidgroup optionally being substituted with one or more substituents R_(x),—C₁-C₁₂ alkylene-(C₅-C₁₄ heterocyclo)-wherein said heterocyclo group maybe a saturated or unsaturated group having one or more rings andcomprising at least one oxygen, nitrogen or sulphur atom in saidring(s), said group optionally being substituted with one or moresubstituents R_(x), —(C₅-C₁₄ heterocyclo)-C₁-C₁₂ alkylene-, wherein saidheterocyclo group may be a saturated or unsaturated group having one ormore rings and comprising at least one oxygen, nitrogen or sulphur atomin said ring(s), said group optionally being substituted with one ormore substituents R_(x), —(OCH₂CH₂)_(r)— and —CH₂—(OCH₂CH₂)_(r)—,wherein each of the above alkylene substituents whether alone orattached to another moiety the carbon chain may optionally besubstituted by one or more substituents R_(x); R₂₀ is a C₁-C₁₂ alkyl oran aryl group having from 6 to 18 carbon atoms in one or more aromaticrings, said aryl groups optionally being substituted with one or moresubstituents R_(x); r is an integer ranging from 1-10; b is an integerof 0 or 1; g is an integer of 0 or 1; w is an integer ranging from 0 to12; each of D, R_(x), X, T, and AA is as defined in any one of claims 1to
 71. 73. A compound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ accordingto claim 72, wherein: L₁ is a linker of formula:

wherein: the wavy line indicates the point of covalent attachment to(T)_(g) if any, or (AA)_(w) if any, or to (X)_(b) if any, or to D; R₁₉is selected from —C₁-C₁₂ alkylene-, —O—(C₁-C₁₂ alkylene), —C₆-C₁₂arylene in one or more rings which may optionally be substituted withone or more substituents R_(x), —C₁-C₁₂ alkylene-C₆-C₁₂ arylene- whereinthe arylene group is in one or more rings which may optionally besubstituted with one or more substituents R_(x), —C₆-C₁₂ arylene-C₁-C₁₂alkylene- wherein the arylene group is in one or more rings which mayoptionally be substituted with one or more substituents R_(x), —C₅-C₁₂heterocyclo- wherein said heterocyclo group may be a saturated orunsaturated group having one or more rings and comprising at least oneoxygen, nitrogen or sulphur atom in said ring(s), said group optionallybeing substituted with one or more substituents R_(x), —C₁-C₁₂alkylene-(C₅-C₁₂ heterocyclo)-wherein said heterocyclo group may be asaturated or unsaturated group having one or more rings and comprisingat least one oxygen, nitrogen or sulphur atom in said ring(s), saidgroup optionally being substituted with one or more substituents R_(x),—(C₅-C₁₂ heterocyclo)-C₁-C₁₂ alkylene- wherein said heterocyclo groupmay be a saturated or unsaturated group having one or more rings andcomprising at least one oxygen, nitrogen or sulphur atom in saidring(s), said group optionally being substituted with one or moresubstituents R_(x), —(OCH₂CH₂)_(r)— and —CH₂—(OCH₂CH₂)_(r)—, whereineach of the above alkylene substituents whether alone or attached toanother moiety the carbon chain may optionally be substituted by one ormore substituents R_(x); r is an integer ranging from 1-6; b is aninteger of 0 or 1; g is an integer of 0 or 1; w is an integer rangingfrom 0 to 12; each of D, R_(x), X, AA, and T is as defined in any one ofclaims 1 to
 71. 74. A compound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁according to claim 72 wherein: L₁ is linker of formula:

wherein: the wavy line indicates the point of covalent attachment to(T)_(g) if any, or (AA)_(w) if any, or to (X)_(b) if any, or to D; R₁₉is selected from —C₁-C₈ alkylene-, —O—(C₁-C₈ alkylene), —C₁-C₈alkylene-C₆-C₁₂ arylene-wherein the arylene group is in one or morerings which may optionally be substituted with one or more substituentsR_(x),and —C₆-C₁₂ arylene-C₁-C₈ alkylene- wherein the arylene group isin one or more rings which may optionally be substituted with one ormore substituents R_(x), wherein each of the above alkylene substituentswhether alone or attached to another moiety the carbon chain mayoptionally be substituted by one or more substituents R_(x); (AA)_(w) isof formula (II):

wherein the wavy lines indicate the point of covalent attachments to(X)_(b) if any, or D (the wavy line to the left) and to (T)_(g) if any,or L₁ or to a hydrogen atom (the wavy line to the right); wherein R₂₁ isselected, at each occurrence, from the group consisting of hydrogen,methyl, isopropyl, sec-butyl, benzyl, indolylmethyl, —(CH₂)₃NHCONH₂,—(CH₂)₄NH₂, —(CH₂)₃NHC(═NH)NH₂ and —(CH₂)₄NHC—(=NH)NH₂, and w is aninteger from 0 to 6; X is an extending group selected from the groupconsisting of where D is conjugated via an amine group (for examplewhere Z is —NH—): —COO—(C₂-C₄ alkylene)NH—, —COO—CH₂-phenylene-NH,wherein said phenylene group may optionally be substituted with from oneto four substituents R_(x) selected from the group consisting of alkylgroups having from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6carbon atoms, halogen atoms, nitro groups and cyano groups, —COO—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —COO—(C₂-C₄ alkylene)S—, and—COO—(C₂-C₄ alkylene)-NHCO(C₁-C₃ alkylene)S—, or where D is conjugatedvia an hydroxy group (for example where Z is —O—): —CONH—(C₂-C₄alkylene)NH—, —COO—CH₂-phenylene-NH—, wherein said phenylene group mayoptionally be substituted with from one to four substituents R_(x)selected from the group consisting of alkyl groups having from 1 to 6carbon atoms, alkoxy groups having from 1 to 6 carbon atoms, halogenatoms, nitro groups and cyano groups, —CONH—(C₂-C₄alkylene)NH—COO—CH₂-(phenylene which may optionally be substituted withfrom one to four substituents R_(x) selected from the group consistingof alkyl groups having from 1 to 6 carbon atoms, alkoxy groups havingfrom 1 to 6 carbon atoms, halogen atoms, nitro groups and cyanogroups)-NH—, —COCH₂NH—COCH₂—NH—, —CONH—(C₂-C₄ alkylene)S—, and—CONH—(C₂-C₄ alkylene)NHCO(C₁-C₃ alkylene)S—; T is an extending groupselected from —CO—(C₁-C₄ alkylene)-NH—; —CO—(C₁-C₄ alkylene)-[O—(C₂-C₄alkylene)]_(j)-NH— and —COO—(C₁-C₄ alkylene)-[O—(C₂-C₄alkylene)]_(j)-NH—, where j is an integer from 1 to 10; b is 0 or 1; gis 0 or 1; and D is a drug moiety of formula (Ia) or a formula (Ib), ora pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof:

or D is a drug moiety of formula (IHa) or a formula (IHb), or apharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof

wherein the wavy lines of (IHa) and (IHb) indicate the point of covalentattachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g) if any,or to L₁; R₁ is —OH or —CN; R₂ is a —C(═O)R_(a) group, wherein R_(a) isselected from hydrogen and substituted or unsubstituted C₁-C₆ alkyl,wherein the optional substituents are one or more substituents R_(x); R₃is hydrogen or a —OR_(b) group wherein R_(b) is a substituted orunsubstituted C₁-C₆ alkyl group, wherein the optional substituents areone or more substituents R_(x); Y is —NH— or —O—; R₄ (if present) is—CH₂OH, or —CH₂NH₂; and Z (if present) is —NH— or —O—.
 75. A compound offormula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ according to claim 72 wherein: L₁is a group of formula:

wherein: the wavy line indicates the point of covalent attachment to(T)_(g) if any, or (AA)_(w) if any, or to (X)_(b) if any or to D; R₁₉ isselected from —C₁-C₆ alkylene-, phenylene-C₁-C₆ alkylene- wherein thephenylene group may optionally be substituted with one or moresubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, halogen atoms, nitro groups and cyano groups, wherein each of theabove alkylene substituents whether alone or attached to another moietyin the carbon chain may optionally be substituted by one or moresubstituents R_(x) selected from the group consisting of alkyl groupshaving from 1 to 6 carbon atoms, alkoxy groups having from 1 to 6 carbonatoms, aryl groups having from 6 to 12 carbon atoms, halogen atoms,nitro groups and cyano groups, and preferably R₁₉ is a C₁-C₆ alkylenegroup; w is 0 or 2, and where w is 2, then (AA)_(w) is of formula (III):

wherein the wavy lines indicate the point of covalent attachments to(X)_(b) if any, or D (the wavy line to the left) and to (T)_(g) if any,or L₁ or to a hydrogen atom (the wavy line to the right); R₂₂ isselected from methyl, benzyl, isopropyl, sec-butyl and indolylmethyl;R₂₃ is selected from methyl, —(CH₂)₄NH₂, —(CH₂)₃NHCONH₂ and—(CH₂)₃NHC(═NH)NH₂; X is an extending group selected from where D isconjugated via an amine group (for example where Z is —NH—):—COO—CH₂-phenylene-NH, —COO(CH₂)₃)NHCOO—CH₂-phenylene-NH,—COO—(CH₂)₃NH—, —COO(CH₂)₃-S-, and —COO—(CH₂)₃NHCO—(CH₂)₂S—; or where Dis conjugated via an hydroxy group (for example where Z is —O—):—COO—CH₂-phenylene-NH—, —CONH(CH₂)₃NHCOOCH₂-phenylene-NH—,—CONH(CH₂)₃NH—, —CONH(CH₂)₃—S—, and —CONH(CH₂)₃NHCO(CH₂)₂S—; wherein Tis an extending group selected from —CO—(C₁-C₄ alkylene)-NH—, —CO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, where j is an integer from 1 to5; b is an integer of 0 or 1; g is 0 or 1; and D is a drug moiety offormula (Ia) or a formula (Ib), or a pharmaceutically acceptable salt,ester, solvate, tautomer or stereoisomer thereof:

or D is a drug moiety of formula (IHa) or a formula (IHb), or apharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof:

wherein the wavy lines of (IHa) and (IHb) indicate the point of covalentattachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g) if any,or to L₁; R₁ is CN or OH; R₂ is acetyl; R₃ is hydrogen or methoxy,preferably hydrogen; Y is —NH— or —O—; R₄ (if present) is —CH₂OH, or—CH₂NH₂; and Z (if present) is —NH— or —O—.
 76. A compound of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ according to claim 72 wherein: L₁ is alinker of formula:

wherein: the wavy line indicates the point of covalent attachment to(T)_(g) if any, or (AA)_(w) if any, or to (X)_(b), if any or to (D); R₁₉is —C₂-C₆ alkylene-; w is 0 or 2, and where w is 2, then (AA)_(w) is offormula (III):

R₂₂ is isopropyl, R₂₃ is selected to methyl and —(CH₂)₃NHCONH₂, whereinthe wavy lines indicate the point of covalent attachments to (X)_(b) ifany, or D (the wavy line to the left) and to (T)_(g) if any, or L₁ or toa hydrogen atom (the wavy line to the right); X is an extending groupselected from —COO—CH₂-phenylene-NH—, —COO(CH₂)₃NHCOO—CH₂-phenylene-NH,—COO—(CH₂)₃)NH—, —COO(CH₂)₃—S—, and —COO—(CH₂)₃NHCO—(CH₂)₂S—; wherein Tis an extending group selected from —CO—(C₁-C₄ alkylene)-NH—, —CO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)-NH—, and —COO—(C₁-C₄alkylene)-[O—(C₂-C₄ alkylene)]_(j)—NH—, where j is an integer from 1 to5; b is 0 or 1; g is 0 or 1; and D is a drug selected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof; wherein the wavy line indicates the point ofcovalent attachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g)if any, or to L₁.
 77. A compound of formulaD-(X)_(b)-(AA)_(w)-(T)_(g)-L₁ according to claim 72 wherein: L₁ is agroup of formula:

wherein: the wavy line indicates the point of covalent attachment to(T)_(g) if any, or (AA)_(w) if any, or to (X)_(b) if any, or to D; R₁₉is-C₂-C₅ alkylene-; w is 0 or 2, and where w is 2, then (AA)_(w) is offormula (III):

wherein R₂₂ is isopropyl, R₂₃ is selected from methyl and—(CH₂)₃NHCONH₂, wherein the wavy lines indicate the point of covalentattachments to (X)_(b) if any, or to D (the wavy line to the left) andto (T)_(g) if any, or L₁ or to a hydrogen atom (the wavy line to theright); X is a —COO—CH₂-phenylene-NH— group; T is a—CO—(CH₂)₂-[O—(CH₂)₂]₄—NH— group; b is an integer of 0 or 1; g is 0 or1; and D is a drug moiety selected from:

or a pharmaceutically acceptable salt, ester, solvate, tautomer orstereoisomer thereof; wherein the wavy line indicates the point ofcovalent attachment to (X)_(b) if any, or (AA)_(w) if any, or to (T)_(g)if any, or to L₁.
 78. A compound of formula D-X-(AA)_(w)-(T)_(g)-L₁according to any one of claims 72 to 77 selected from:


79. A compound of formula D-X-(AA)_(w)-(T)_(g)-L₁ according to any oneof claims 72 to 77 selected from:


80. A compound of formula D-(X)_(b)-(AA)_(w)-(T)_(g)-L₁, wherein each ofD, X, AA, T, L₁, b, g and w are as defined in any one of claims 1 to 70.81. A drug conjugate according to any one of claims 1 to 71, or acompound according to any one of claims 72 to 80, wherein b+g+w is not0.
 82. A drug conjugate according to any one of claims 1 to 71 or 81, ora compound according to any one of claims 72 to 81, wherein b+w is not0.
 83. A drug conjugate according to any one of claims 1 to 71, 81 or82, or a compound according to any one of claims 72 to 82, wherein whenw is not 0, then b is
 1. 84. Use of a drug moiety as described in anyone of claims 1 to 23, as a payload in an antibody drug conjugate. 85.Use of a drug moiety as described in any one of claims 1 to 23, in themanufacture of a antibody drug conjugate.
 86. A drug conjugate accordingto any one of claims 1 to 71, for use as a medicament.
 87. A drugconjugate according to any one of claims 1 to 71 for use in thetreatment of cancer, and more preferably a cancer selected from lungcancer, colorectal cancer, breast cancer, pancreas carcinoma, kidneycancer, leukaemia, multiple myeloma, lymphoma, gastric and ovariancancer.
 88. A pharmaceutical composition comprising a drug conjugateaccording to any one of claims 1 to 71 and a pharmaceutically acceptablecarrier.
 89. A method for the prevention or treatment of cancercomprising administering an effective amount of a drug conjugateaccording to any one of claims 1 to 71 to a patient in need thereof. 90.A method for the treatment of cancer according to claim 89, wherein thecancer is selected from lung cancer, colorectal cancer, breast cancer,pancreas carcinoma, kidney cancer, leukaemia, multiple myeloma,lymphoma, gastric and ovarian cancer.
 91. Use of a drug conjugateaccording to any one of claims 1 to 71 in the preparation of amedicament for the treatment of cancer, preferably a cancer selectedfrom lung cancer, colorectal cancer, breast cancer, pancreas carcinoma,kidney cancer, leukaemia, multiple myeloma, lymphoma, gastric andovarian cancer.
 92. A kit comprising a therapeutically effective amountof a drug conjugate according to any one of claims 1 to 71 and apharmaceutically acceptable carrier.
 93. The kit according to claim 92for use in the treatment of cancer, and more preferably a cancerselected from lung cancer, colorectal cancer, breast cancer, pancreascarcinoma, kidney cancer, leukaemia, multiple myeloma, lymphoma, gastricand ovarian cancer.
 94. The drug conjugate according to any one ofclaims 1 to 50, wherein n is in the range of from 1-12, 1-8, 3-8, 3-6,3-5, 1, 2, 3, 4, 5 or 6; preferably 3, 4 or 5 or more preferably
 4. 95.A process for the preparation of a drug antibody conjugate according toany one of claims 1 to 71 comprising conjugating a moiety Ab comprisingat least one antigen binding site and a drug D, Ab and D being asdefined in any one of claims 1 to
 71. 96. A process according to claim95 for the preparation of a drug antibody conjugate of formula (G) or(G′):

said process comprising the following steps: (i) reacting a drug D-H offormula (IH)-H:

wherein the substituents in the definitions of (IH)-H are as defined inany one of claims 1 to 71, with a compound of formula (D′) or (E):

to give a compound of formula (F) or (F′),respectively:

(ii) partial reduction of one or more disulfide bonds in the antibody tobe conjugated to give a reduced antibody Ab-SH having free thiol groups:

and (iii) reaction of the partially reduced antibody Ab-SH having freethiol groups with the compound of formula (F) or (F′) produced in step(i) to give the desired drug antibody conjugate of formula (G) or (G′)respectively:


97. A process according to claim 96, wherein the antibody is selectedfrom Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab,an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and ananti-CD 30 antibody, or an antigen-binding fragment or animmunologically active portion thereof which is partially reduced instep (ii) using tris[2-carboxyethyl]phosphine hydrochloride, preferablythe antibody is selected from Trastuzumab and an anti-CD13 antibody oran antigen-binding fragment or an immunologically active portion thereofwhich is partially reduced in step (ii) usingtris[2-carboxyethyl]phosphine hydrochloride and most preferably it isTrastuzumab or an antigen-binding fragment or an immunologically activeportion thereof which is partially reduced in step (ii) usingtris[2-carboxyethyl]phosphine hydrochloride.
 98. A process according toclaim 95 for the preparation of a drug antibody conjugate of formula (W)or (W′):

said process comprising the following steps: (i) reacting the antibodywith 2-iminothiolane hydrochloride (Traut's reagent) to give athiol-activated antibody:

(ii) reacting the thiol-activated antibody with the compound of formula(F) or (F′), to give the desired drug antibody conjugate of formula (W)or (W′), respectively.


99. A process according to claim 98, wherein the antibody is selectedfrom Brentuximab, Gemtuzumab, Inozutumab, Rovalpituzumab, Trastuzumab,an anti-CD4 antibody, an anti-CD5 antibody, an anti-CD13 antibody and ananti-CD 30 antibody, or an antigen-binding fragment or animmunologically active portion thereof, preferably the antibody isselected from Trastuzumab and an anti-CD13 antibody or anantigen-binding fragment or an immunologically active portion thereofand most preferably it is Trastuzumab or an antigen-binding fragment oran immunologically active portion thereof.
 100. A process according toclaim 95 for the preparation of a drug antibody conjugate of formula (O)or (P):

said process comprising the following steps: (i) either: (a) reacting adrug (D-H) of formula formula (IH)-H:

wherein the substituents in the definitions of (IH)-H are as defined inany one of claims 1 to 71, with a compound of formula X₂-C(O)-X₁ whereinX₁ and X₂ are leaving groups to give a compound of formula (B):

and the point of attachment of the —(C═O)X₁ moiety is the free —NH₂group of the compound of formula D-H, or (b) reacting said drug (D-H) offormula (IH)-H as defined above with 4-nitro-phenylchloroformate to givea compound of formula (J):

and the point of attachment of the (4-nitrophenyl)-O—CO— group is thesame as that for the X₁(CO) moiety in (a) above; (ii) either: (c)reacting the compound of formula (B) produced in step (i) with a hydroxycompound of formula HO—(CH₂)₁₋₆NHProt^(NH) and removing the Prot^(NH)group from the coupled compound to give a compound of formula (C):

and then reacting the resulting compound of formula (C) with a compoundof formula Me-S-S—(CH₂)₁₋₃—CO₂H to give a compound of formula (K)

or (d) reacting the compound (J) produced in step (i) with a compound offormula HO—(CH₂)₁₋₃SProt^(SH) and removing the Prot^(SH) group from thecoupled compound to give a compound of formula (L):

(iii) reacting (K) or (L) produced in step (ii) with dithiothreitolunder disulfide reducing conditions to give compounds of formula (M) and(N) respectively:

(iv) reacting the antibody to be conjugated withsuccininimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate toderivatise said antibody at one or more lysine groups with asuccininimidyl-4-(N-maleimidomethyl)cyclohexane-1-carbonyl group:

(v) reacting the derivatised antibody produced in step (iv) with either(M) or (N) produced in step (iii) to give the desired drug antibodyconjugate of formula (O) or (P):


101. A process according to claim 100, wherein the compound of formulaX₂-C(O)-X₁ is 1,1′-carbonyldiimidazole.
 102. A process according toclaim 100 or claim 101, wherein the hydroxy compound reacted with thecompound of formula (B) is preferably HO—(CH₂)₂₋₄—NHProtNH, andpreferably HO—(CH₂)₃—NHProt^(NH).
 103. A process according to claim 100or claim 101, wherein the compound reacted with the compound of formula(C) to give the compound of formula (K) is 3-(methyldisulfanyl)propanoicacid.
 104. A process according to claim 100, wherein the compound offormula HO—(CH₂)₁₋₃SProt^(SH) that is reacted with a compound of formula(J) to give a compound of formula (L) is HO—(CH₂)₃SProt^(SH).